Methods for screening voltage-gated proteins

ABSTRACT

In one aspect, the invention relates to a method for identifying a compound which modulates the activity of a voltage-gated protein. In certain embodiments, the voltage gate protein is a voltage-gated ion channel. In certain embodiments, the voltage-gated protein is a voltage sensitive phosphatase. In certain embodiments, the voltage-gated protein used in conjunction with the methods of the invention is modified to altered permeability or voltage sensitivity.

This application is a Divisional of U.S. application Ser. No. 14/473,386filed Aug. 29, 2014, which is a Continuation-In-Part of InternationalPatent Application No. PCT/US2013/028324 filed Feb. 28, 2013, whichclaims priority of U.S. Provisional Patent Application No. 61/604,897,filed Feb. 29, 2012, the contents of which are hereby incorporated byreference in their entirety.

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosureas it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

All patents, patent applications and publications cited herein arehereby incorporated by reference in their entirety. The disclosures ofthese publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art as known to those skilled therein as of the date of theinvention described herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 5, 2016, isnamed 2206689.121US3_SL.txt and is 864,760 bytes in size.

BACKGROUND OF THE INVENTION

Voltage-gated ion channels are important targets for therapeuticintervention. Defects in voltage-gated ion channel function are linkedto numerous biological outcomes, including, but not limited tocardiovascular, metabolic, and autoimmune disorders, pain andneurological disorders, and cancer. The identification of molecules thatmodulate the activity of voltage-gated proteins (hereafter, “VGPs”) isdifficult and relatively non-specific. There is a demand for screeningmethods suitable for identifying molecules that bind specifically toparticular VGPs functional regions and for identifying molecules thatbind specifically to particular VGP functional/conformational states.This invention addresses these needs.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a method for identifying acompound which modulates the activity of a voltage-gated ion channel,the method comprising (a) providing a voltage-gated ion channel in astructure that separates a first medium from a second medium, whereinthe voltage-gated ion channel exhibits independent ion permeation, (b)contacting the voltage-gated ion channel with a test compound, (c)measuring the amount of said independent ion permeation through thevoltage-gated ion channel between the first and second media, and (d)comparing the amount of said independent ion permeation measured for thevoltage-gated ion channel contacted with the test compound to the amountof said independent ion permeation measured for the voltage-gated ionchannel not contacted with the test compound, wherein an increase ordecrease in the amount of said independent ion permeation of thevoltage-gated ion channel contacted with the test compound compared tothe voltage-gated ion channel not contacted with the test compoundindicates that the test compound modulates the activity of thevoltage-gated ion channel.

In certain embodiments, the voltage-gated ion channel comprises one ormore amino acid mutations that cause the voltage gate ion channel toexhibit alpha pore independent permeation.

In one aspect, the invention relates to a method for identifying acompound which modulates the activity of a voltage-sensitivephosphatase, the method comprising (a) providing a voltage-sensitivephosphatase in a structure that separates a first medium from a secondmedium, (b) contacting the voltage-sensitive phosphatase with a testcompound, (c) measuring the activity of the voltage-sensitivephosphatase, and (d) comparing the amount of said activity measured forthe voltage-sensitive phosphatase contacted with the test compound tothe amount of said activity measured for the voltage-sensitivephosphatase not contacted with the test compound, wherein an increase ordecrease in the amount of activity of the voltage-sensitive phosphatasecontacted with the test compound compared to the voltage-sensitivephosphatase not contacted with the test compound indicates that the testcompound modulates the activity of the voltage-sensitive phosphatase.

In certain embodiments, the voltage sensitive phosphatase comprises oneor more amino acid mutations that cause the voltage sensitivephosphatase to exhibit altered voltage sensitivity.

In certain embodiments the structure that separates a first medium froma second medium is polarized. In certain embodiments, the degreestructure polarization is varied. In certain embodiments, the polarizedstructure has a voltage difference of about −300 mV to about +300 mV. Incertain embodiments, the structure is a lipid bilayer. In certainembodiments, the structure is a liposome membrane. In certainembodiments, the structure comprises a naturally occurring membrane, asynthetic membrane, or any combination thereof.

In certain embodiments, the structure is a cellular membrane of a cell.

In certain embodiments, the cell is an animal cell, a plant cell, afungal cell, a yeast cell, a bacterial cell, or an archaebacterial cell.In certain embodiments, the cell is an oocyte, a fibroblast, anepithelial cell, or a myocyte. In certain embodiments, the cell is acell from a cell line.

In certain embodiments, the cellular membrane is in a cell. In certainembodiments, the cellular membrane is in a permeabilized cell. Incertain embodiments, the cellular membrane is not in a cell. In certainembodiments, the cellular membrane comprises an extracellular membrane,an intracellular membrane, a vesicular membrane, an organelle membrane,or any combination thereof.

In certain embodiments, the contacting of step (b) is performed byadding the compound to either the first medium or the second medium. Incertain embodiments, the cellular membrane the contacting of step (b) isperformed by adding the compound to the first medium and the secondmedium. In certain embodiments, the method further comprises a step ofcontacting the voltage-gated ion channel with one or more ion channelmodulating agents before step (b).

In certain embodiments, the one or more ion channel modulating agent isselected from the group comprising a turret blocking agent, a main-poreblocking agent, a gating-modifying agent, a cysteine-tethered reagent,or a voltage sensing domain toxin.

In certain embodiments, the voltage-gated ion channel is a protonchannel, a sodium channel, a potassium channel, a calcium channel, or avoltage activated enzyme.

In certain embodiments, measuring the amount of independent ionpermeation through the voltage-gated ion channel between the first andsecond media is by patch-clamp measurement. In certain embodiments,measuring the amount of independent ion permeation through thevoltage-gated ion channel between the first and second media is byfluorescence measurement. In certain embodiments, measuring the amountof independent ion permeation through the voltage-gated ion channelbetween the first and second media is by radiolabeled measurement. Incertain embodiments, measuring the amount of independent ion permeationthrough the voltage-gated ion channel between the first and second mediais by biological assay measurement.

In certain embodiments, the independent ion permeation is omega-poredependent ion permeation. In certain embodiments, the independent ionpermeation is sigma-pore dependent ion permeation.

In certain embodiments, the one or more of the amino acid mutations arein one or more voltage sensing domains of the voltage-gated ion channelor the voltage sensitive phosphatase. In certain embodiments, the one ormore of the amino acid mutations are S4 helix mutations. In certainembodiments, the one or more of the amino acid mutations are not S4helix mutations. In certain embodiments, at least one of the amino acidmutations is an S4 helix mutation and at least one of the amino acidmutations is not an S4 helix mutation.

In certain embodiments, the method further comprises a step ofcontacting the voltage-gated ion channel with MTSET or MTSES before step(b). In certain embodiments, the method further comprises a step ofcontacting the voltage sensitive phosphatase with MTSET or MTSES beforestep (b).

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows the structural basis of Voltage Sensing Domain (VSD)channelopathies and that VSD gating charge mutations cause pathologicalcation leaks. Resting state conformations of wild type (left) and R2Sermutant (right) VSDs are shown. Cations selectively leak through themutant VSDs when the gating charge mutation is proximal to Phe233(labeled F233).

FIG. 2 shows that the Kv1.2 and Kv1.3 pore domains maintain stablestructures during molecular dynamics simulations at depolarizingpotential. The root-mean-square deviation (RMSD) of the pore domain CAatoms (all four (identical) subunits that compose the tetrameric pore)is plotted versus simulation time.

FIG. 3 shows K⁺ permeation for Kv1.2 and Kv1.3. Cumulative K⁺ permeationevents through the pore domain, at depolarizing potential, are plottedversus simulation time.

FIG. 4 shows gating charge displacement of Kv1.3 compared with the Kv1.2resting state as reference. The Kv1.2 resting state gating chargedisplacement is shown at as grey line. The Kv1.3 gating chargedisplacement (red: depolarizing [control] potential; green: depolarizingpotential with bound ShK toxin; blue: hyperpolarizing potential) isplotted versus simulation time. “C27” refers to the molecular dynamicsforce field used; the number of atoms in each simulation (“107 k”, etc.)are indicated.

FIG. 5 shows the structure of the ShK/Kv1.3 complex. In this “ribbon”representation, based on the three-dimensional coordinates of the CAatoms, the pore domain of Kv1.3 is grey; the core of each VSD is green;the mobile S3b and S4 portions of each VSD are red; remaining portionsof each VSD are light blue; and the ShK toxin is purple. ShK is bound tothe pore at hyperpolarzing and weak depolarizing potentials, and blocksthe ion permeation pathway from the extracellular side.

FIGS. 6A-D show a contact map of ShK and Kv1.3 interacting residues.Data are plotted separately for each subunit (FIG. 6A, FIG. 6B, FIG. 6C,and FIG. 6D) of the pore domain. Interactions (within 4 Å distance)between particular Kv1.3 residues (horizontal axis) and particular ShKresidues (vertical axis) is indicated by the white-to-red color scale(red representing more contact). Green dots correspond to experimentallyderived nuclear magnetic resonance data (Mol. Pharmacol. 75(4) 762-773(2009); Mol. Pharmacol. 67(4) 1369-1381 (2005); JBC 274(31) 21885-21892(1999); JBC 273(49) 32697-32707(1998); BBRC 219(3) 696-701 (1996))

FIG. 7 shows that large depolarizing potentials separate Shk from theKv1.3 pore. In the left panel, the positions of permeating K+(top; ionscolored distinctly) and the ammonium group of ShK residue Lys22 (bottom)are plotted versus simulation time; the z coordinate is relative to thecenter of the membrane in which Kv1.3 is embedded. The K+ occupancy ofthe “S0 site” in the pore domain “selectivity filter” (“SF”; z˜=7.5 Å)and the position of the ShK Lys22 ammonium group are correlated. Oncethe Sext and S0 sites are unoccupied by K+, the toxin can reach the poredomain “turret” insert a positive side chain (e.g., the Lys22 ammoniumgroup) into the selectivity filter, thus occluding the S0 site. S0 is50% occupied by K+, and Sext is presumably occupied even less. As thechannel enters the non-conducting resting state (hyperpolarizingpotentials), the ShK binding preferentially involves binding to stateswith S0 unoccupied. The on-rate increases with stronger hyperpolarizingvoltage. The ShK off-rate increases with increased depolarizing voltage.

FIG. 8 shows the ShK toxin bound to the Kv1.3 pore. The Kv1.3 poredomain is shown as a green ribbon; ShK is shown as a molecular surfacerepresentation (mostly black), in which negatively charged residues arecolored red and positively charged residues are colored blue. The purplehighlight shows the ShK (positively charged) Lys22 bound at the Kv1.3 SF51 site. Two different time snapshots, displaying two of severalpossible poses, from the simulation are shown.

FIG. 9 indicates whether particular small molecule ligands, bound to theextracellular vestibule of a KV1Kv1.2/KV2.1 VSD, remain bound to the VSDor escape. Columns in this table, from left to right, are the ligandidentifier, the VSD identifier, the ligand force field used, and whetherthe ligand escaped (and at what time).

FIG. 10 indicates whether particular small molecule ligands, bound tothe extracellular vestibule of a Kv1.2 VSD, remain bound to the VSD orescape. Columns in this table, from left to right, are the ligandidentifier, the VSD (and simulation run) identifier, the transmembranepotential, and whether the ligand escaped.

FIG. 11 shows GxTx-1E toxin bound in one of several possible bindingmodes, to the Kv1.2/Kv2.1 chimera VSD. The toxin is shown as a surfacerepresentation, with the aromatic residues colored yellow and the otherresidues colored blue. The VSD is shown as a cyan-colored ribbonrepresentation, with the loop connecting S3b to S4 (residues 274-284)colored orange. The membrane lipids are shown as stick figures (coloredcyan [carbon], blue [nitrogen], and red [oxygen and phosphorus]); thephosphate phosphorus atoms are highlighted as red spheres.

FIG. 12 shows a contact map of GxTx-1E and Kv1.2/Kv2.1 chimera VSDinteracting residues. Data are plotted separately for the VSD S3-S4(left) and S1-S2 (right) loops. Interactions between particularKv1.2/Kv2.1 residues (horizontal axis) and particular GxTx-1E residues(vertical axis) is indicated by the white-to-red color scale (redrepresenting more contact).

FIG. 13 shows a binding depth profile GxTx-1E/Kv1.2/2.1.

FIGS. 14A-AM show the amino acid sequences of various VGICs, includingDrosophila Shaker (GI:288442) (SEQ ID NO: 1), human Na_(v) 1.1 (HGNC:SCN1A) (SWISS-PROT: P35498) (SEQ ID NO: 2), human Na_(v) 1.2 (HGNC:SCN2A) (SWISS-PROT: Q99250) (SEQ ID NO: 3), human Na_(v) 1.3 (HGNC:SCN3A) (SWISS-PROT: Q9NY46) (SEQ ID NO: 4), human Na_(v) 1.4 (HGNC:SCN4A) (GENBANK TRANSLATION: M81758) (SEQ ID NO: 5), human Na_(v) 1.5(HGNC: SCN5a) (SWISS-PROT: Q14524) (SEQ ID NO: 6), human Na_(v) 1.6(HGNC: SCN8A) (SWISS-PROT: 095788) (SEQ ID NO: 7), human Na_(v) 1.7(HGNC: SCN9A) (GENBANK TRANSLATION: X82835) (SEQ ID NO: 8), human Na_(v)1.8 (HGNC: SCN10A) (SWISS-PROT: Q9Y5Y9) (SEQ ID NO: 9), human Na_(v) 1.9(HGNC: SCN11A) (SWISS-PROT: Q9UHE0) (SEQ ID NO: 10), human Ca_(v)1.1(HGNC: CACNA1S) (GENBANK TRANSLATION: L33798) (SEQ ID NO: 11), humanCa_(v)1.2 (HGNC: CACNA1C) (GENBANK TRANSLATION: L29529) (SEQ ID NO: 12),human Ca_(v)1.3 (HGNC: CACNA1D) (GENBANK TRANSLATION: M76558) (SEQ IDNO: 13), human Ca_(v)1.4 (HGNC: CACNA1F) (GENBANK TRANSLATION: AJ224874)(SEQ ID NO: 14), human Ca_(v)2.1 (HGNC: CACNA1A) (GENBANK TRANSLATION:AF004883) (SEQ ID NO: 15), human Ca_(v)2.2 (HGNC: CACN1B) (GENBANKTRANSLATION: M94172) (SEQ ID NO: 16), human Ca_(v)2.3 (HGNC: CACNA1E)(GENBANK TRANSLATION: L29384) (SEQ ID NO: 17), human Ca_(v)3.1 (HGNC:CACNA1G) (SWISS-PROT: 043497) (SEQ ID NO: 18), human Ca_(v)3.2 (HGNC:CACNA1H) (SWISS-PROT: 095180) (SEQ ID NO: 19), human Ca_(v)3.3 (HGNC:CACNA11) (GENBANK: AAM67414) (SEQ ID NO: 20), human K_(v)1.1 (NCBI:KCNA1) (NCBI: NM_000217) (SEQ ID NO: 21), human K_(v)1.2 (HGNC: KCNA2)(NCBI: NM_004974) (SEQ ID NO: 22), human K_(v)1.3 (HGNC: KCNA3) (NCBI:NM_002232) (SEQ ID NO: 23), human K_(v)1.4 (HGNC: KCNA4) (NCBI:NM_002233) (SEQ ID NO: 24), human K_(v)1.5 (HGNC: KCNA5) (NCBI:NM_002234) (SEQ ID NO: 25), human K_(v)1.6 (HGNC: KCNA6) (NCBI:NM_002235) (SEQ ID NO: 26), human K_(v)1.7 (HGNC: KCNA7) (NCBI:NM_031886) (SEQ ID NO: 27), human K_(v)1.8 (HGNC: KCNA10) (NCBI:NM_005549) (SEQ ID NO: 28), human K_(v)2.1 (HGNC: KCNB1) (NCBI:NM_004975) (SEQ ID NO: 29), human K_(v)2.2 (HGNC: KCNB2) (NCBI:NM_004770) (SEQ ID NO: 30), human K_(v)3.1 (HGNC: KCNC1) (NCBI:NM_004976) (SEQ ID NO: 31), human K_(v)3.2 (HGNC: KCNC2) (NCBI:NM_139136) (SEQ ID NO: 32), human K_(v)3.3 (HGNC: KCNC3) (NCBI:NM_004977) (SEQ ID NO: 33), human K_(v)3.4 (HGNC: KCNC4) (NCBI:NM_004978) (SEQ ID NO: 34), human K_(v)4.1 (HGNC: KCND1) (NCBI:NM_004979) (SEQ ID NO: 35), human K_(v)4.2 (HGNC: KCND2) (NCBI:NM_012281) (SEQ ID NO: 36), human K_(v)4.3 (HGNC: KCND3) (NCBI:NM_004980) (SEQ ID NO: 37), human K_(v)5.1 (HGNC: KCNF1) (NCBI:NM_002236) (SEQ ID NO: 38), human K_(v)6.1 (HGNC: KCNG1) (NCBI:NM_002237) (SEQ ID NO: 39), human K_(v)6.2 (HGNC: KCNG2) (NCBI:NM_012283) (SEQ ID NO: 40), human K_(v)6.3 (HGNC: KCNG3,) (NCBI:NM_133329) (SEQ ID NO: 41), human K_(v)6.4 (HGNC: KCNG4,) (NCBI:NM_172347) (SEQ ID NO: 42), human K_(v)7.1 (HGNC: KCNQ1,) (NCBI:NM_000218) (SEQ ID NO: 43), human K_(v)7.2 (HGNC: KCNA2,) (NCBI:NM_172107) (SEQ ID NO: 44), human K_(v)7.3 (HGNC: KCNA3,) (NCBI:NM_004519) (SEQ ID NO: 45), human K_(v)7.4 (HGNC: KCNA4) (NCBI:NM_004700) (SEQ ID NO: 46), human K_(v)7.5 (HGNC: KCNQ) (NCBI:NM_019842) (SEQ ID NO: 47), human K_(v)8.1 (HGNC: KCNV1,) (NCBI:NM_014379) (SEQ ID NO: 48), human K_(v)8.2 (HGNC: KCNV2,) (NCBI:NM_133497) (SEQ ID NO: 49), human K_(v)9.1 (HGNC: KCNS1,) (NCBI:NM_002251) (SEQ ID NO: 50), human K_(v)9.2 (HGNC: KCNS2,) (NCBI:NM_020697) (SEQ ID NO: 51), human K_(v)9.3 (HGNC: KCNS3) (NCBI:NM_023966) (SEQ ID NO: 52), human K_(v)10.1 (HGNC: KCNH1) (NCBI:NM_172362) (SEQ ID NO: 53), human K_(v)10.2 (HGNC: KCNH5) (NCBI:NM_139318) (SEQ ID NO: 54), human K_(v)11.1 (HGNC: KCNH2) (NCBI:NM_000238) (SEQ ID NO: 55), human K_(v)11.2 (HGNC: KCNH6) (NCBI:NM_030779) (SEQ ID NO: 56), human K_(v)11.3 (HGNC: KCNH7) (NCBI:NM_033272) (SEQ ID NO: 57), human K_(v)12.1 (HGNC: KCNH8) (NCBI:NM_144633) (SEQ ID NO: 58), human K_(v)12.2 (HGNC: KCNH3) (NCBI:NM_012284) (SEQ ID NO: 59), human K_(v)12.3 (HGNC: KCNH4) (NCBI:NM_012285) (SEQ ID NO: 60), human HCN1 (HGNC: HCN1) (NCBI: NM_021072)(SEQ ID NO: 61), human HCN2 (HGNC: HCN2) (NCBI: NM_001194) (SEQ ID NO:62), human HCN3 (HGNC: HCN3) (NCBI: NM_020897) (SEQ ID NO: 63), humanHCN4 (HGNC: HCN4) (NCBI: NM_005477) (SEQ ID NO: 64), human CatSper1(HGNC: CatSper1) (GENBANK TRANSLATION: AF407333) (SEQ ID NO: 65), humanCatSper2 (HGNC: None) (GENBANK TRANSLATION: AF411817) (SEQ ID NO: 66),human CatSper3 (HGNC: None) (GENBANK TRANSLATION: AF432876) (SEQ ID NO:67), human CatSper4 (HGNC: None) (GENBANK TRANSLATION: BN000273) (SEQ IDNO: 68), human Hv1 (HGNC: HVCN1) (NCBI: NP 001035196.1) (SEQ ID NO: 69),human K_(Ca)1.1 (HGNC: KCNMA1) (NCBI: NM_001014797) (SEQ ID NO: 70),human K_(Ca)4.1 (HGNC: KCNT1) (NCBI: NM_020822) (SEQ ID NO: 71), humanK_(Ca)4.2 (HGNC: KCNT2) (NCBI: NM_198503) (SEQ ID NO: 72), human TPC1(HGNC: None) (NCBI: NP 001137291.1) (SEQ ID NO: 73),

FIGS. 15A-BU show an amino acid sequence alignment of of various VGICs,including Drosophila Shaker (GI:288442) (SEQ ID NO: 1), human Na_(v) 1.1(HGNC: SCN1A) (SWISS-PROT: P35498) (SEQ ID NO: 2), human Na_(v) 1.2(HGNC: SCN2A) (SWISS-PROT: Q99250) (SEQ ID NO: 3), human Na_(v) 1.3(HGNC: SCN3A) (SWISS-PROT: Q9NY46) (SEQ ID NO: 4), human Na_(v) 1.4(HGNC: SCN4A) (GENBANK TRANSLATION: M81758) (SEQ ID NO: 5), human Na_(v)1.5 (HGNC: SCN5a) (SWISS-PROT: Q14524) (SEQ ID NO: 6), human Na_(v) 1.6(HGNC: SCN8A) (SWISS-PROT: 095788) (SEQ ID NO: 7), human Na_(v) 1.7(HGNC: SCN9A) (GENBANK TRANSLATION: X82835) (SEQ ID NO: 8), human Na_(v)1.8 (HGNC: SCN10A) (SWISS-PROT: Q9Y5Y9) (SEQ ID NO: 9), human Na_(v) 1.9(HGNC: SCN11A) (SWISS-PROT: Q9UHE0) (SEQ ID NO: 10), human Ca_(v)1.1(HGNC: CACNA1S) (GENBANK TRANSLATION: L33798) (SEQ ID NO: 11), humanCa_(v)1.2 (HGNC: CACNA1C) (GENBANK TRANSLATION: L29529) (SEQ ID NO: 12),human Ca_(v)1.3 (HGNC: CACNA1D) (GENBANK TRANSLATION: M76558) (SEQ IDNO: 13), human Ca_(v)1.4 (HGNC: CACNA1F) (GENBANK TRANSLATION: AJ224874)(SEQ ID NO: 14), human Ca_(v)2.1 (HGNC: CACNA1A) (GENBANK TRANSLATION:AF004883) (SEQ ID NO: 15), human Ca_(v)2.2 (HGNC: CACN1B) (GENBANKTRANSLATION: M94172) (SEQ ID NO: 16), human Ca_(v)2.3 (HGNC: CACNA1E)(GENBANK TRANSLATION: L29384) (SEQ ID NO: 17), human Ca_(v)3.1 (HGNC:CACNA1G) (SWISS-PROT: 043497) (SEQ ID NO: 18), human Ca_(v)3.2 (HGNC:CACNA1H) (SWISS-PROT: 095180) (SEQ ID NO: 19), human Ca_(v)3.3 (HGNC:CACNA11) (GENBANK: AAM67414) (SEQ ID NO: 20), human K_(v)1.1 (NCBI:KCNA1) (NCBI: NM_000217) (SEQ ID NO: 21), human K_(v)1.2 (HGNC: KCNA2)(NCBI: NM_004974) (SEQ ID NO: 22), human K_(v)1.3 (HGNC: KCNA3) (NCBI:NM_002232) (SEQ ID NO: 23), human K_(v)1.4 (HGNC: KCNA4) (NCBI:NM_002233) (SEQ ID NO: 24), human K_(v)1.5 (HGNC: KCNA5) (NCBI:NM_002234) (SEQ ID NO: 25), human K_(v)1.6 (HGNC: KCNA6) (NCBI:NM_002235) (SEQ ID NO: 26), human K_(v)1.7 (HGNC: KCNA7) (NCBI:NM_031886) (SEQ ID NO: 27), human K_(v)1.8 (HGNC: KCNA10) (NCBI:NM_005549) (SEQ ID NO: 28), human K_(v)2.1 (HGNC: KCNB1) (NCBI:NM_004975) (SEQ ID NO: 29), human K_(v)2.2 (HGNC: KCNB2) (NCBI:NM_004770) (SEQ ID NO: 30), human K_(v)3.1 (HGNC: KCNC1) (NCBI:NM_004976) (SEQ ID NO: 31), human K_(v)3.2 (HGNC: KCNC2) (NCBI:NM_139136) (SEQ ID NO: 32), human K_(v)3.3 (HGNC: KCNC3) (NCBI:NM_004977) (SEQ ID NO: 33), human K_(v)3.4 (HGNC: KCNC4) (NCBI:NM_004978) (SEQ ID NO: 34), human K_(v)4.1 (HGNC: KCND1) (NCBI:NM_004979) (SEQ ID NO: 35), human K_(v)4.2 (HGNC: KCND2) (NCBI:NM_012281) (SEQ ID NO: 36), human K_(v)4.3 (HGNC: KCND3) (NCBI:NM_004980) (SEQ ID NO: 37), human K_(v)5.1 (HGNC: KCNF1) (NCBI:NM_002236) (SEQ ID NO: 38), human K_(v)6.1 (HGNC: KCNG1) (NCBI:NM_002237) (SEQ ID NO: 39), human K_(v)6.2 (HGNC: KCNG2) (NCBI:NM_012283) (SEQ ID NO: 40), human K_(v)6.3 (HGNC: KCNG3,) (NCBI:NM_133329) (SEQ ID NO: 41), human K_(v)6.4 (HGNC: KCNG4,) (NCBI:NM_172347) (SEQ ID NO: 42), human K_(v)7.1 (HGNC: KCNQ1,) (NCBI:NM_000218) (SEQ ID NO: 43), human K_(v)7.2 (HGNC: KCNA2,) (NCBI:NM_172107) (SEQ ID NO: 44), human K_(v)7.3 (HGNC: KCNA3,) (NCBI:NM_004519) (SEQ ID NO: 45), human K_(v)7.4 (HGNC: KCNA4) (NCBI:NM_004700) (SEQ ID NO: 46), human K_(v)7.5 (HGNC: KCNQ) (NCBI:NM_019842) (SEQ ID NO: 47), human K_(v)8.1 (HGNC: KCNV1,) (NCBI:NM_014379) (SEQ ID NO: 48), human K_(v)8.2 (HGNC: KCNV2,) (NCBI:NM_133497) (SEQ ID NO: 49), human K_(v)9.1 (HGNC: KCNS1,) (NCBI:NM_002251) (SEQ ID NO: 50), human K_(v)9.2 (HGNC: KCNS2,) (NCBI:NM_020697) (SEQ ID NO: 51), human K_(v)9.3 (HGNC: KCNS3) (NCBI:NM_023966) (SEQ ID NO: 52), human K_(v)10.1 (HGNC: KCNH1) (NCBI:NM_172362) (SEQ ID NO: 53), human K_(v)10.2 (HGNC: KCNH5) (NCBI:NM_139318) (SEQ ID NO: 54), human K_(v)11.1 (HGNC: KCNH2) (NCBI:NM_000238) (SEQ ID NO: 55), human K_(v)11.2 (HGNC: KCNH6) (NCBI:NM_030779) (SEQ ID NO: 56), human K_(v)11.3 (HGNC: KCNH7) (NCBI:NM_033272) (SEQ ID NO: 57), human K_(v)12.1 (HGNC: KCNH8) (NCBI:NM_144633) (SEQ ID NO: 58), human K_(v)12.2 (HGNC: KCNH3) (NCBI:NM_012284) (SEQ ID NO: 59), human K_(v)12.3 (HGNC: KCNH4) (NCBI:NM_012285) (SEQ ID NO: 60), human HCN1 (HGNC: HCN1) (NCBI: NM_021072)(SEQ ID NO: 61), human HCN2 (HGNC: HCN2) (NCBI: NM_001194) (SEQ ID NO:62), human HCN3 (HGNC: HCN3) (NCBI: NM_020897) (SEQ ID NO: 63), humanHCN4 (HGNC: HCN4) (NCBI: NM_005477) (SEQ ID NO: 64), human CatSper1(HGNC: CatSper1) (GENBANK TRANSLATION: AF407333) (SEQ ID NO: 65), humanCatSper2 (HGNC: None) (GENBANK TRANSLATION: AF411817) (SEQ ID NO: 66),human CatSper3 (HGNC: None) (GENBANK TRANSLATION: AF432876) (SEQ ID NO:67), human CatSper4 (HGNC: None) (GENBANK TRANSLATION: BN000273) (SEQ IDNO: 68), human Hv1 (HGNC: HVCN1) (NCBI: NP 001035196.1) (SEQ ID NO: 69),human K_(Ca)1.1 (HGNC: KCNMA1) (NCBI: NM_001014797) (SEQ ID NO: 70),human K_(Ca)4.1 (HGNC: KCNT1) (NCBI: NM_020822) (SEQ ID NO: 71), humanK_(Ca)4.2 (HGNC: KCNT2) (NCBI: NM_198503) (SEQ ID NO: 72), human TPC1(HGNC: None) (NCBI: NP 001137291.1) (SEQ ID NO: 73). The alignment wasgenerated using the Clustal W2 multiple sequence alignment toolaccording to the following parameters: (a) Protein Wieght Matrix:BLOSUM; (b) Gap Open: 5; (c) Gap Extension: 0.1; (d) Gap Distances: 5;(e) End Gaps; (f) No iteration; (g) Numiter: 1; (h) Clustering: NJ.

FIGS. 16A-D show the activated-state-to-resting-state transition of avoltage-gated K+ channel. Membrane-lateral (FIG. 16A, FIG. 16B) andintracellular (FIG. 16C, FIG. 16D) views of activated (FIG. 16A, FIG.16C; open, conducting) and resting (FIG. 16B, FIG. 16D; closed,non-conducting) states of KV1.2/KV2.1; two or four subunits are shown,along with K+ ions (green) and water molecules (red/white) in theselectivity filter (SF). The graph indicates the number of pore cavitywater molecules (grey surface). Magnified views in b and d illustratewater-filled and empty pore cavities in activated and resting states(hydrophobic constriction at the conserved Pro-Val-Pro motif [purple]).Results from simulations either with (“T1+”) or without (“T1−”) thefunctionally nonessential cytoplasmic T1 domain, separated by dashedlines, are both shown in d. In the resting state, helix S6 is lockedinto a straight conformation by Leu331 (S5)-Pro405 (S6) side-chaininterchange (M. Ø. Jensen et al., (2010) Proc. Natl. Acad. Sci. USA107:5833-5838). Inset: K+ currents. The currents and H2O/K+ permeationratio (˜1) agree with experiment (H. Ando et al., (2005) J. Gen.Physiol. 126:529-538) and pore-only simulations (M. Ø. Jensen et al.,(2010) Proc. Natl. Acad. Sci. USA 107:5833-5838). VSD-pore separation(right), which increases, for instance, the R1(Q)-Ala351 distance by ˜20Å, explains lack of resting-state inter-domain crosslinking (M. Lainè etal., Neuron 39:467-481). Both states are compatible with Shakertryptophan tolerance mutagenesis data of VSD helices S1-S3a (K. H. Hong& C. Miller, (2000) J. Gen. Physiol. 115:51-58).

FIGS. 17A-D show VSD motion during gating. FIG. 17A: Consecutive VSDconfigurations illustrate sequential inward movement of the S4gating-charge residues during the gating transition; the fourindividually colored traces track the position of their Cα atoms. R2exhibits a Cα displacement of 15.4±2.5 Å, averaged over all fullyrelaxed, resting state VSDs in T1− simulations 2 and 3, and 14.3±0.9 Åfor T1+ (sim. 8), in agreement with KvAP (V. Ruta et al., (2005) Cell123:463-475; A. Banerjee & R. MacKinnon, (2008) J. Mol. Biol.381:569-580) and Shaker (H. P. Larsson et al., (1996) Neuron 16:387-397)accessibility data, and with crosslinking of, for instance, ShakerIle230 (S2) and R1(Q) only in the resting state (F. V. Campos et al.,(2007) Proc. Natl. Acad. Sci. USA 104:7904-7909). FIG. 17B: Local S4helix rotation vs R2-R4 z-positions. FIG. 17C: Cumulative VSD gatingcharge displacements, Q(t)=Σi qi [f(zi,t)−f(zi,0)]. f(z) is thefractional potential drop (blue/white/red background in a) along themembrane normal, z; zi,t is the z-position of VSD atom i at time t, andqi is its partial charge. The gating charge, 13.3±0.4 e, was estimatedas the difference between the final (average) charge displacements atdepolarizing and hyperpolarizing potentials; the gating process wasinitiated at −750 mV, reducing the magnitude to −375 mV at −70 is (sim.3 [see FIGS. 21]) and ˜145 is (sim. 4), and increasing it to −500 mV at−205 is (sim. 8). The contribution of S4 only is explicitly shown(black) for one simulation, revealing that S4 fully accounts for thegating charge displacement. Inset: Fractional potential drop across theVSD, in both activated and resting states, obtained from free energycalculations; gating-charge residue z-positions are also shown. FIG.17D: Cα root-mean-square deviations for the entire channel tetramer, thepore domain (tetramer), and a single VSD decomposed into S1-S3a (loopsomitted), S3b-S4 (the “paddle”), and S4 alone. Inset: Schematic of thefull channel.

FIGS. 18A-H show key steps of resting-state-to-activated-statetransition of a voltage-gated K+ channel. Deactivation (red) and earlyreactivation (blue; first ˜100 μs) upon depolarization (FIG. 18A, FIG.18B): inward and outward movement of S4 gating-charge residues and“paddle” Cα RMSD relative to the initial structure (FIG. 18A), and totalgating charge transfer (FIG. 18B). Late reactivation (c-h; final ˜10 μs,after essentially all gating charge has been transferred): pore cavityrewetting, Leu331 (S5)-Pro405 (S6) side-chain interchange, andcumulative outward K+ permeation events (FIG. 18C); K+ population ofpore cavity (FIG. 18D) and SF (FIG. 18E); S4-S5 linker/helix S6interaction energies (FIG. 18F); S4-S5 linker/helix S6 contacts (FIG.18G); and upper gate (Ile402/site S5) lateral opening (FIG. 18H).

FIG. 19 shows a mechanistic model for voltage gating. Subjecting theactivated state (1) to hyperpolarizing voltage initiates S4 inwardmovement and VSD-pore lateral separation. Ion depletion of the porecavity (2)—coupled to inward motion of a single S4—leads to porehydrophobic collapse: closure of the upper (Ile402) and lower gates [PVPmotif; Leu331 (S5)-Pro405 (S6) side-chain interchange (M. Ø. Jensen etal., (2010) Principles of conduction and hydrophobic gating in K+channels. Proc. Natl. Acad. Sci. USA 107:5833-5838)] halts conduction(3). S4 continues inward and VSD-pore separation increases; as S4completes its inward motion, the S4-S5 linker helix moves fully down andthe VSDs separate from the pore, consolidating the resting state (4).Subjecting the resting state to depolarizing voltage drives S4 outward.When all four S4 and S4-S5 linker helices are fully up (5), and all VSDshave repacked against the pore, the lower gate becomes destabilized; the4→5 transition constitutes the rate-limiting step in the activationprocess. Lower gate fluctuation triggers pore opening and partial porerehydration—water molecules cooperatively enter the cavity—that allowion entry and initial outward conduction (6); the 5→6 transition isessentially voltage-independent. The presence of ions drives completepore rehydration, which in turn fully opens the upper and lower gatesand, again in an essentially voltage-independent manner, returns thechannel to its activated state (1). The lateral position of the VSDs(circles) relative to the pore domain (squares) is shown schematically(extracellular view).

FIGS. 20A-C show the “omega” pore. Resting state conformations of wildtype (FIG. 20A) and R2Ser mutant (FIG. 20C) VSDs. Spheres in (FIG. 20A)mark residues accessible to chemical modification from the extracellular(yellow) and intracellular (purple) sides in the resting state (H. P.Larsson et al., (1996) Transmembrane movement of the Shaker K⁺ channelS4. Neuron 16:387-397), consistent with ˜15-Å S4 inward motion [see FIG.17a ]. FIG. 20B Water and K⁺ (R2Ser) densities (arbitrary units). TheR2Ser mutation increases hydration at Phe233 by ˜50%, facilitating inpart K⁺ permeation. The ion permeation pathway—the omega pore—in theR2Ser mutant is shown as a green mesh in (FIG. 20C). The green surfaceand spheres indicate predominant K⁺“sites” and actual positions from asingle permeation event; the blue surfaces represent VSD hydration.

FIG. 21 shows a summary of simulations. Listed for each simulation arethe force field; presence (+) or absence (−) of the T1 domain; theapproximate number of atoms; the total simulation time; the appliedvoltage (when the voltage was adjusted toward the end of the simulationthe final value is given in parenthesis); the times for pore half- andfull-dewetting (i.e., pore closure); the gating charge at closure; and,at simulation end, the number of gating-charge arginine residues (“Rs”;R-1, R0, R2, R3, R4) in each of the voltage-sensing domains (VSDs;subunits A/B/C/D) that were at, or inward of, Phe233; and the gatingcharge transferred (q_(g)). The time at which the pore closed wasdefined as when the water occupancy of the pore cavity dropped belowfive water molecules; the half-dewetted time was defined as when <25water molecules occupied the cavity. Reactivation simulations 12 and 13started from resting state configurations obtained from simulation 8,either with all VSDs but one fully down (Rs: 3/3/1/3; simulation 12), orwith all VSDs fully down (Rs: 3/3/3/3; simulation 13). Simulation 14 wasa control simulation with the TIP4P water model (SF patch omitted). Thesimulation was stopped at ˜60 μs after observation of dewetting andinitial gating charge movement.

FIG. 22 shows a revised side-chain partial charges. Revised charges foraspartate, glutamate, and arginine side-chain atoms, denoted by “DER”and “DER2” The corresponding original CHARMM27 (“C27”) charges are alsoshown.

FIG. 23 shows omega pore simulations. Two mutants—R2Ser and R0Asn—weresimulated with or without an additional glutamate-to-aspartate mutationat position 226. Listed for each simulation are the voltage; simulationtime (time used for analysis); inward K⁺ leak current; and averagegating charge. The analysis time was typically shorter than the totalsimulated time because the isolated VSDs were unstable over longertimescales at |V|>750 mV; at approximately ˜400 mV, however, the VSDswere stable for longer than 100 μs. Calculation of the averagecumulative charge displacements indicates that the gating charge of bothmutants is ˜2 e per subunit.

FIGS. 24A-B show double bilayer simulations. FIG. 24A. The transmembranepotential through the VSD-centered on the R4 side chain for theactivated state, and the R2 side chain for the resting state—wascomputed as the difference between the electrostatic potentials from thecharge-imbalanced and charge-balanced double bilayer simulations(simulation times are given in parentheses), using the VMD PMEPOT plugin(A. Aksimentiev, K. Schulten, Imaging α-hemolysin with moleculardynamics: Ionic conductance, osmotic permeability and the electrostaticpotential map. Biophys. J. 88, 3745-3761 (2005)) interfaced to HiMach(T. Tu et al., A scalable parallel framework for analyzing terascalemolecular dynamics simulation trajectories. Proceedings of the ACM/IEEEConference on Supercomputing (SC08) (ACM Press, New York, 2008)). Foreach state subject to neutral or hyperpolarizing potentials, averagepotential profiles along the z-axis through the VSD were computed as anaverage over the (time-averaged) profiles lying within 1.6 Å radius (inthe xy-plane) from the VSD center. The charge difference leads to atransmembrane potential difference of 558±1.4 mV computed from theactivated state double bilayer trajectory as the difference in betweenelectrostatic potentials in the “inner” and “outer” compartments. FIG.24 B: Outward and inward K+ currents (simulations 1 and 5).

FIGS. 25A-E show protein—lipid interactions. FIG. 25A: Upper panel:lipid exposure for VSD S1-S3a residues, in activated and resting states,compared to experimental Trp-substitution tolerance data (K. H. Hong, C.Miller, The lipid-protein interface of a Shaker K⁺ channel. J. Gen.Physiol. 115, 51-58 (2000)). FIG. 25B: Resting-to-activated statedifference in exposure. FIG. 25C: Resting-to-activated state differencefor VSD S5-S6 residues. FIG. 25D: Individual S4 gating-charge residuecoordination to lipid phosphate groups in activated (A) and resting (R)states, using the first 10 and last 30 μs of simulations 5 and 6 at ahyperpolarizing voltage. For comparison, coordination to the activatedstate at a depolarizing voltage (simulation 1) is also shown. FIG. 25E:Snapshots of lipid coordination to R0 in the activated state (left) andto R-1, R0, R2, R4 and K5 in the resting state (right).

FIG. 26 shows potential drop across the voltage-sensing domain.Potential drops are depicted as a fraction of the total potential drop(V) for activated (“A”) and resting (“R”) state conformations of thevoltage-sensing domain. Potentials were obtained from free energycalculations with a single sensor in a single bilayer, where V wasimposed through a constant electric field, and from double bilayersimulations with two anti-parallel-oriented VSDs, one in each bilayer,where V was imposed through charge imbalance [see FIG. 27]; AU,arbitrary units. Only data for one VSD from the double bilayersimulations are shown; the data for the other VSD are the same withinerror. The fractional potential drop, f(z), was fit tof(z)=1/[exp(−c(z−z′))+1]. For both states, the constant electric fieldand the charge imbalance methods give similar results. Notably, thefield is strongest at R4, in accord with the observations of the gatingprocess described herein: R4 is always the first S4 arginine to moveinward. The positional distributions (of Arg C_(ζ) and Lys N_(ζ)) fromthe equilibrium simulation of a single VSD are shown for reference.

FIG. 27 shows water occupancy of the pore cavity. The water occupancy isshown as a function of time for simulations 1-8 and 14 [see FIG. 21]. Afully hydrated cavity holds about 40 water molecules.

FIG. 28 shows Ion selectivity in the “omega” pore. In this resting-stateconformation of the R2Ser mutant voltage sensing domain (VSD) the cationpermeation pathway—the omega pore—is shown as a grey mesh; the greensurface indicates high-occupancy “sites” during K+ permeation throughthe omega pore. The discontinuous purple surfaces represent Cl− density,obtained from the same simulation. The Cl− density, which is contouredat the same level as the continuous K+ density (grey mesh), indicates noanion permeation across the VSD center.

FIGS. 29A-R show amino acid sequences for the voltage sensing domains ofHomo_hvcn1 gi_91992153_(SEQ ID NO: 74); Gallus_hvcn gi_71897219_(SEQ IDNO: 75); opossum_hvcn gi_12632423_(SEQ ID NO: 76); 004 rat_hvcn1gi_109497399_(SEQ ID NO: 77); Equus_hvcn1 gi_194214323_(SEQ ID NO: 78);bos_hvcn1 gi_119909285_(SEQ ID NO: 79); sus_hvcn1 gi_194042948_(SEQ IDNO: 80); Macaca_hvcn_4 gi_109098724_(SEQ ID NO: 81); Macaca_hvcngi_109098722_(SEQ ID NO: 82); Macaca_hvcn_2 gi_10909872_(SEQ ID NO: 83);dog_hvcn1 2 gi_73994604_(SEQ ID NO: 84); dog_hvcn1 gi_73994606_(SEQ IDNO: 85); mus_hvcn1 gi_109809757_(SEQ ID NO: 86); Xenopus_t_hvcn1gi_58332220_(SEQ ID NO: 87); Xenopus_1_hvcn1 gi_148235789_148235(SEQ IDNO: 88); Danio_hvcn1 gi_50539752_(SEQ ID NO: 89); tetraodon_hvcn1gi_47209646_(SEQ ID NO: 90); takifugu_hvcn1 ENSEMBL UPI00016E3E8E(SEQ IDNO: 91); nematostella_hvcn gi_156364735_(SEQ ID NO: 92); Ciona_hvcngi_118344228_(SEQ ID NO: 93); trichoplex_hvcn1 gi_196002093_(SEQ ID NO:94); human_CACNA1E_repeat_3 gi_53832005_(SEQ ID NO: 95);Drosophila_CAC1A_repeat_3 gi_24641459_(SEQ ID NO: 96);mouse_CAC1H_repeat_1 gi_254826786_(SEQ ID NO: 97); Homo_CAC1I_repeat_1gi_51093859_(SEQ ID NO: 98); Homo_CAC1G_repeat3 sp_O43497(SEQ ID NO:99); Gallus_SCN1A_repeat1 uniprot_E1C4S3(SEQ ID NO: 100);rat_SCN2A_repeat1 sp_P04775(SEQ ID NO: 101); mouse_SCN1A_repeat1uniprot_A2APX8(SEQ ID NO: 102); mouse_SCN1A_repeat1 uniprot_A2APX7(SEQID NO: 103); rat_SCN11A_repeat1 sp_O88457(SEQ ID NO: 104);mouse_SCN11A_repeat1 sp_Q9R053(SEQ ID NO: 105); Homo_SCN11A_repeat1sp_Q9UI33(SEQ ID NO: 106); taeniopygia_SCN_repeat1 gi_224044620 (SEQ IDNO: 107); Homo_SCN4A_repeat1 sp_P35499(SEQ ID NO: 199);rat_SCN5A_repeat1 sp_P15389(SEQ ID NO: 108); rat_SCN9A_repeat1sp_O08562(SEQ ID NO: 109); rabbit_SCN9A_repeat1 sp_Q28644(SEQ ID NO:110); Homo_SNC3A_repeat1 sp_Q9NY46(SEQ ID NO: 111); Canis_SCN_repeat1gi_74004456 (SEQ ID NO: 112); Danio_SCN8AA_repeat1 sp_Q9DF53(SEQ ID NO:113); mouse_SCN8A_repeat1 sp_Q9WTU3(SEQ ID NO: 114); Canis_SCNAA_repeat1sp_O46669(SEQ ID NO: 115); Homo_SCN7A_repeat1 sp_Q01118(SEQ ID NO: 116);rabbit_CAC1C_repeat1 sp_P15381(SEQ ID NO: 117); mouse_CAC1S_repeat1sp_Q02789(SEQ ID NO: 118); mouse_CAC1F_repeat1 sp_Q9JIS7(SEQ ID NO:119); Gallus_CAC1D_repeat1 sp 073700(SEQ ID NO: 120); Homo_CACN_repeat1gi_193788728 (SEQ ID NO: 121); Drosophila_CAC1D_repeat1 sp_Q24270(SEQ IDNO: 122); Homo_CAC1A_repeat1 sp_O00555(SEQ ID NO: 123);Homo_CAC1B_repeat1 sp_Q00975(SEQ ID NO: 124); rat_SCN11A_repeat3sp_O88457(SEQ ID NO: 125); mouse_SCN11A_repeat3 sp_Q9R053(SEQ ID NO:126); rat_SCN9A_repeat3 sp_O08562(SEQ ID NO: 127); rabbit_SCN9A_repeat3sp_Q28644(SEQ ID NO: 128); mouse_SCN9A_repeat3 uniprot_B7ZWN(SEQ ID NO:129); mouse_KCNH1 sp_Q60603(SEQ ID NO: 130); mouse_KCNH8 sp_P59111(SEQID NO: 131); Homo_KCNH3 sp_Q9ULD8(SEQ ID NO: 132); Homo_CAC1G_repeat4sp_O43497(SEQ ID NO: 133); mouse_SCN11A_repeat4 sp_Q9R053(SEQ ID NO:134); rat_SCN9A_repeat4 sp_O08562(SEQ ID NO: 135); rat_SCN11A_repeat4sp_O88457(SEQ ID NO: 136); humo_CAC1G_repeat2 sp_O43497(SEQ ID NO: 137);Homo_CACNA1E_repeat_4 sp_Q15878(SEQ ID NO: 138);Drosophila_CAC1A_repeat_4 sp_P91645(SEQ ID NO: 139); Homo_KCNV2sp_Q8TDN2(SEQ ID NO: 140); Homo_KCNF1 sp_Q9H3M0_KCNF1(SEQ ID NO: 141);Homo_KCNB1 sp_Q14721(SEQ ID NO: 142); Canis_KCNB2 sp_Q95167(SEQ ID NO:143); Drosophila_KCNAB sp_P17970(SEQ ID NO: 144); pongo_KCNV1sp_Q5RC10(SEQ ID NO: 145); Homo_KCNS3 sp_Q9BQ31(SEQ ID NO: 146);squirrelmonkey_KCNS1 sp A4K2X4(SEQ ID NO: 147); Gallus_KCNG2sp_O73606(SEQ ID NO: 148); Homo_KCNG4 sp_Q8TDN1(SEQ ID NO: 149);rat_KCNC3 sp_Q01956_KCNC3(SEQ ID NO: 150); Homo_KCNC2 sp_Q96PR1(SEQ IDNO: 151); drosophila KCNAW sp_P17972(SEQ ID NO: 152); Homo_KCNA1sp_Q09470(SEQ ID NO: 153); rat KNCA6 sp_P17659(SEQ ID NO: 154);Homo_KCNA5 sp_P22460(SEQ ID NO: 155); rat_KCNA3 sp_P15384(SEQ ID NO:156); Canis_Kv1.3 gi_57088651_(SEQ ID NO: 157); bovine_KCNA4sp_Q05037(SEQ ID NO: 158); Homo_KCA10 sp_Q16322(SEQ ID NO: 159);rat_Kv1.2 2R9R_b_vs gi_16087779_(SEQ ID NO: 160); Homo_Kvgi_4826782_(SEQ ID NO: 161); rat_Kv pdb:2A79_chainb (SEQ ID NO: 162);Canis_KCNA2 sp_Q28293(SEQ ID NO: 163); Drosophila_shaker_Kchannelgi_288442_(SEQ ID NO: 164); rabbit_KCND3 sp_Q9TTT5(SEQ ID NO: 165);hum_CACNA1E_repeat_2 sp_Q15878(SEQ ID NO: 166);Drosophila_CAC1A_repeat_2 sp_P91645(SEQ ID NO: 167);mouse_SCN11A_repeat2 sp_Q9R053(SEQ ID NO: 168); rat_SCN11A_repeat2sp_O88457(SEQ ID NO: 169); rat_SCN9A_repeat2 sp_O08562(SEQ ID NO: 170);ornitho_C15orf27_gi_149410687_(SEQ ID NO: 171); Danio_c15orf27gi_123703002_(SEQ ID NO: 172); monodelphis_C15orf27 gi_12627230_(SEQ IDNO: 173); sus_C15orf27 gi_194039682 (SEQ ID NO: 174); Homo_C15orf27gi_118442841_(SEQ ID NO: 175); pan_C15orf27_gi_114658268_(SEQ ID NO:176); horse_C15orf27 gi_149692210_(SEQ ID NO: 177); mus_C15orf27gi_27370422_(SEQ ID NO: 178); rat_C15or27 gi_157817759_(SEQ ID NO: 179);Ciona_C15orf gi_198433556 (SEQ ID NO: 180); Methanococcus_hyperpol_Kvsp_Q57603(SEQ ID NO: 181); ornitho_vsp gi_149635858_(SEQ ID NO: 182);Xenopus_t_vsp gi_62859843_(SEQ ID NO: 183); Gallus_vsp gi_118084924_(SEQID NO: 184); Danio_vsp gi_70887553_(SEQ ID NO: 185); Xenopus_vspgi_148230800_(SEQ ID NO: 186); rat_vsp gi_157820295_(SEQ ID NO: 187);mus_vsp gi_40549440_(SEQ ID NO: 188); dog_vsp gi_73993164_(SEQ ID NO:189); human_vsp gi_213972591_(SEQ ID NO: 190); Homo_vsp_gammagi_40549435_(SEQ ID NO: 191); Ciona_vsp gi_76253898_(SEQ ID NO: 192);Aeropyrum_Kv PDB_1ORS_c(SEQ ID NO: 201); Homo_BK gi_119574982_(SEQ IDNO: 193); and mouse_BK_mslo gi_4639628_(SEQ ID NO: 194).

FIGS. 30A-J show a multiple alignment of the voltage sensing domainamino acid sequences. Designation 001 is the voltage sensing domain ofHomo_hvcn1 gi_91992153_(SEQ ID NO: 74); designation 002 is the voltagesensing domain of Gallus_hvcn gi_71897219_(SEQ ID NO: 75); designation003 is the voltage sensing domain of opossum_hvcn gi_12632423_(SEQ IDNO: 76); designation 004 is the voltage sensing domain of rat_hvcn1gi_109497399_(SEQ ID NO: 77); designation 005 is the voltage sensingdomain of Equus_hvcn1 gi_194214323_(SEQ ID NO: 78); designation 006 isthe voltage sensing domain of bos_hvcn1 gi_119909285_(SEQ ID NO: 79);designation 007 is the voltage sensing domain of sus_hvcn1gi_194042948_(SEQ ID NO: 80); designation 008 is the voltage sensingdomain of Macaca_hvcn_4 gi_109098724_(SEQ ID NO: 81); designation 009 isthe voltage sensing domain of Macaca_hvcn gi_109098722_(SEQ ID NO: 82);designation 010 is the voltage sensing domain of Macaca_hvcn_2gi_10909872_(SEQ ID NO: 83); designation 011 is the voltage sensingdomain of dog_hvcn1_2 gi_73994604_(SEQ ID NO: 84); designation 012 isthe voltage sensing domain of dog_hvcn1 gi_73994606_(SEQ ID NO: 85);designation 013 is the voltage sensing domain of mus_hvcn1gi_109809757_(SEQ ID NO: 86); designation 014 is the voltage sensingdomain of Xenopus_t_hvcn1 gi_58332220_(SEQ ID NO: 87); designation 015is the voltage sensing domain of Xenopus_1_hvcn1 gi_148235789 148235(SEQID NO: 88); designation 016 is the voltage sensing domain of Danio_hvcn1gi_50539752_(SEQ ID NO: 89); designation 017 is the voltage sensingdomain of tetraodon_hvcn1 gi_47209646_(SEQ ID NO: 90); designation 018is the voltage sensing domain of takifugu_hvcn1 ENSEMBLUPI00016E3E8E(SEQ ID NO: 91); designation 019 is the voltage sensingdomain of nematostella_hvcn gi_156364735_(SEQ ID NO: 92); designation020 is the voltage sensing domain of Ciona_hvcn gi_118344228_(SEQ ID NO:93); designation 021 is the voltage sensing domain of trichoplex_hvcn1gi_196002093_(SEQ ID NO: 94); designation 022 is the voltage sensingdomain of human_CACNA1E_repeat_3 gi_53832005_(SEQ ID NO: 95);designation 023 is the voltage sensing domain ofDrosophila_CAC1A_repeat_3 gi_24641459_(SEQ ID NO: 96); designation 024is the voltage sensing domain of mouse_CAC1H_repeat_1 gi_254826786_(SEQID NO: 97); designation 025 is the voltage sensing domain ofHomo_CAC1I_repeat_1 gi_51093859_(SEQ ID NO: 98); designation 026 is thevoltage sensing domain of Homo_CAC1G_repeat3 sp_O43497(SEQ ID NO: 99);designation 027 Gallus_SCN1A_repeat1 uniprot_E1C4S3(SEQ ID NO: 100);designation 028 is the voltage sensing domain of rat_SCN2A_repeat1sp_P04775(SEQ ID NO: 101); designation 029 is the voltage sensing domainof mouse_SCN1A_repeat1 uniprot_A2APX8(SEQ ID NO: 102); designation 030is the voltage sensing domain of mouse_SCN1A_repeat1 uniprot_A2APX7(SEQID NO: 103); designation 031 is the voltage sensing domain ofrat_SCN11A_repeat1 sp_O88457(SEQ ID NO: 104); designation 032 is thevoltage sensing domain of mouse_SCN11A_repeat1 sp_Q9R053(SEQ ID NO:105); designation 033 is the voltage sensing domain ofHomo_SCN11A_repeat1 sp_Q9UI33(SEQ ID NO: 106); designation 034 is thevoltage sensing domain of taeniopygia_SCN_repeat1 gi_224044620_(SEQ IDNO: 107); designation 035 is the voltage sensing domain ofHomo_SCN4A_repeat1 sp_P35499(SEQ ID NO: 199); designation 036 is thevoltage sensing domain of rat_SCN5A_repeat1 sp_P15389(SEQ ID NO: 108);designation 037 is the voltage sensing domain of rat_SCN9A_repeat1sp_O08562(SEQ ID NO: 109); designation 038 is the voltage sensing domainof rabbit_SCN9A_repeat1 sp_Q28644(SEQ ID NO: 110); designation 039 isthe voltage sensing domain of Homo_SNC3A_repeat1 sp_Q9NY46(SEQ ID NO:111); designation 040 is the voltage sensing domain of Canis_SCN_repeat1gi_74004456_(SEQ ID NO: 112); designation 041 is the voltage sensingdomain of Danio_SCN8AA_repeat1 sp_Q9DF53(SEQ ID NO: 113); designation042 is the voltage sensing domain of mouse_SCN8A_repeat1 sp_Q9WTU3(SEQID NO: 114); designation 043 is the voltage sensing domain ofCanis_SCNAA_repeat1 sp_O46669(SEQ ID NO: 115); designation 044 is thevoltage sensing domain of Homo_SCN7A_repeat1 sp_Q01118(SEQ ID NO: 116);designation 045 is the voltage sensing domain of rabbit_CAC1C_repeat1sp_P15381(SEQ ID NO: 117); designation 046 is the voltage sensing domainof mouse_CAC1S_repeat1 sp_Q02789(SEQ ID NO: 118); designation 047 is thevoltage sensing domain of mouse_CAC1F_repeat1 sp_Q9JIS7(SEQ ID NO: 119);designation 048 is the voltage sensing domain of Gallus_CAC1D_repeat1 sp073700(SEQ ID NO: 120); designation 049 is the voltage sensing domain ofHomo_CACN_repeat1 gi_193788728_(SEQ ID NO: 121); designation 050 is thevoltage sensing domain of Drosophila_CAC1D_repeat1 sp_Q24270(SEQ ID NO:122); designation 051 is the voltage sensing domain ofHomo_CAC1A_repeat1 sp_O00555(SEQ ID NO: 123); designation 052 is thevoltage sensing domain of Homo_CAC1B_repeat1 sp_Q00975(SEQ ID NO: 124);designation 053 is the voltage sensing domain of rat_SCN11A_repeat3sp_O88457(SEQ ID NO: 125); designation 054 is the voltage sensing domainof mouse_SCN11A_repeat3 sp_Q9R053(SEQ ID NO: 126); designation 055 isthe voltage sensing domain of rat_SCN9A_repeat3 sp_O08562(SEQ ID NO:127); designation 056 is the voltage sensing domain ofrabbit_SCN9A_repeat3 sp_Q28644(SEQ ID NO: 128); designation 057 is thevoltage sensing domain of mouse_SCN9A_repeat3 uniprot_B7ZWN(SEQ ID NO:129); designation 058 is the voltage sensing domain of mouse_KCNH1sp_Q60603(SEQ ID NO: 130); designation 059 is the voltage sensing domainof mouse_KCNH8 sp_P59111(SEQ ID NO: 131); designation 060 is the voltagesensing domain of Homo_KCNH3 sp_Q9ULD8(SEQ ID NO: 132); designation 061is the voltage sensing domain of Homo_CAC1G_repeat4 sp_O43497(SEQ ID NO:133); designation 062 is the voltage sensing domain ofmouse_SCN11A_repeat4 sp_Q9R053(SEQ ID NO: 134); designation 063 is thevoltage sensing domain of rat_SCN9A_repeat4 sp_O08562(SEQ ID NO: 135);designation 064 is the voltage sensing domain of rat_SCN11A_repeat4sp_O88457(SEQ ID NO: 136); designation 065 is the voltage sensing domainof humo_CAC1G_repeat2 sp_O43497(SEQ ID NO: 137); designation 066 is thevoltage sensing domain of Homo_CACNA1E_repeat_4 sp_Q15878(SEQ ID NO:138); designation 067 is the voltage sensing domain ofDrosophila_CAC1A_repeat_4 sp_P91645(SEQ ID NO: 139); designation 068 isthe voltage sensing domain of Homo_KCNV2 sp_Q8TDN2(SEQ ID NO: 140);designation 069 is the voltage sensing domain of Homo_KCNF1sp_Q9H3M0_KCNF1(SEQ ID NO: 141); designation 070 is the voltage sensingdomain of Homo_KCNB1 sp_Q14721(SEQ ID NO: 142); designation 071 is thevoltage sensing domain of Canis_KCNB2 sp_Q95167(SEQ ID NO: 143);designation 072 is the voltage sensing domain of Drosophila_KCNABsp_P17970(SEQ ID NO: 144); designation 073 is the voltage sensing domainof pongo_KCNV1 sp_Q5RC10(SEQ ID NO: 145); designation 074 is the voltagesensing domain of Homo_KCNS3 sp_Q9BQ31(SEQ ID NO: 146); designation 075is the voltage sensing domain of squirrelmonkey_KCNS1 sp A4K2X4(SEQ IDNO: 147); designation 076 is the voltage sensing domain of Gallus_KCNG2sp_O73606(SEQ ID NO: 148); designation 077 is the voltage sensing domainof Homo_KCNG4 sp_Q8TDN1(SEQ ID NO: 149); designation 078 is the voltagesensing domain of rat_KCNC3 sp_Q01956_KCNC3(SEQ ID NO: 150); designation079 is the voltage sensing domain of Homo_KCNC2 sp_Q96PR1(SEQ ID NO:151); designation 080 is the voltage sensing domain of Drosophila_KCNAWsp_P17972(SEQ ID NO: 152); designation 081 is the voltage sensing domainof Homo_KCNA1 sp_Q09470(SEQ ID NO: 153); designation 082 is the voltagesensing domain of rat KNCA6 sp_P17659(SEQ ID NO: 154); designation 083is the voltage sensing domain of Homo_KCNA5 sp_P22460(SEQ ID NO: 155);designation 084 is the voltage sensing domain of rat_KCNA3 sp_P15384(SEQID NO: 156); designation 085 is the voltage sensing domain ofCanis_Kv1.3 gi_57088651_(SEQ ID NO: 157); designation 086 is the voltagesensing domain of bovine_KCNA4 sp_Q05037(SEQ ID NO: 158); designation087 is the voltage sensing domain of Homo_KCA10 sp_Q16322(SEQ ID NO:159); designation 088 is the voltage sensing domain of rat_Kv1.22R9R_b_vs gi_16087779_(SEQ ID NO: 160); designation 089 is the voltagesensing domain of Homo_Kv gi_4826782_(SEQ ID NO: 161); designation 090is the voltage sensing domain of rat_Kv pdb:2A79_chainb(SEQ ID NO: 162);designation 091 is the voltage sensing domain of Canis_KCNA2sp_Q28293(SEQ ID NO: 163); designation 092 is the voltage sensing domainof Drosophila_shaker_Kchannel gi_288442_(SEQ ID NO: 164); designation093 rabbit_KCND3 sp_Q9TTT5(SEQ ID NO: 165); designation 094 is thevoltage sensing domain of hum_CACNA1E_repeat_2 sp_Q15878(SEQ ID NO:166); designation 095 is the voltage sensing domain ofDrosophila_CAC1A_repeat_2 sp_P91645(SEQ ID NO: 167); designation 096 isthe voltage sensing domain of mouse_SCN11A_repeat2 sp_Q9R053(SEQ ID NO:168); designation 097 is the voltage sensing domain ofrat_SCN11A_repeat2 sp_O88457(SEQ ID NO: 169); designation 098 is thevoltage sensing domain of rat_SCN9A_repeat2 sp_O08562(SEQ ID NO: 170);designation 099 is the voltage sensing domain ofornitho_C15orf27_gi_149410687 (SEQ ID NO: 171); designation 100 is thevoltage sensing domain of Danio_c15orf27 gi_123703002_(SEQ ID NO: 172);designation 101 is the voltage sensing domain of monodelphis_C15orf27gi_12627230_(SEQ ID NO: 173); designation 102 is the voltage sensingdomain of sus_C15orf27 gi_194039682_(SEQ ID NO: 174); designation 103 isthe voltage sensing domain of Homo_C15orf27 gi_118442841_(SEQ ID NO:175); designation 104 is the voltage sensing domain ofpan_C15orf27_gi_114658268_(SEQ ID NO: 176); designation 105 is thevoltage sensing domain of horse_C15orf27_gi_149692210_(SEQ ID NO: 177);designation 106 is the voltage sensing domain of mus_C15orf27gi_27370422_(SEQ ID NO: 178); designation 107 is the voltage sensingdomain of rat_C15or27 gi_157817759_(SEQ ID NO: 179); designation 108 isthe voltage sensing domain of Ciona_C15orf gi_198433556_(SEQ ID NO:180); designation 109 is the voltage sensing domain ofMethanococcus_hyperpol_Kv sp_Q57603(SEQ ID NO: 181); designation 110 isthe voltage sensing domain of ornitho_vsp gi_149635858_(SEQ ID NO: 182);designation 111 is the voltage sensing domain of Xenopus_t_vspgi_62859843_(SEQ ID NO: 183); designation 112 is the voltage sensingdomain of Gallus_vsp gi_118084924_(SEQ ID NO: 184); designation 113 isthe voltage sensing domain of Danio_vsp gi_70887553_(SEQ ID NO: 185);designation 114 is the voltage sensing domain of Xenopus_vspgi_148230800_(SEQ ID NO: 186); designation 115 is the voltage sensingdomain of rat_vsp gi_157820295_(SEQ ID NO: 187); designation 116 is thevoltage sensing domain of mus_vsp gi_40549440_(SEQ ID NO: 188);designation 117 is the voltage sensing domain of dog_vspgi_73993164_(SEQ ID NO: 189); designation 118 is the voltage sensingdomain of human_vsp gi_213972591_(SEQ ID NO: 190); designation 119 isthe voltage sensing domain of Homo_vsp_gamma gi_40549435_(SEQ ID NO:191); designation 120 is the voltage sensing domain of Ciona_vspgi_76253898_(SEQ ID NO: 192); designation 121 is the voltage sensingdomain of Aeropyrum_Kv PDB_1ORS_c(SEQ ID NO: 201); designation 122 isthe voltage sensing domain of Homo_BK gi_119574982_(SEQ ID NO: 193);designation 123 is the voltage sensing domain of mouse_BK_mslogi_4639628_(SEQ ID NO: 194).

FIG. 31 shows the amino acid sequences of CiVSP voltage-sensorcontaining phosphatase [Ciona intestinalis](GenBank: BAD98733.1) (SEQ IDNO. 195); DrVSP voltage-sensing phosphoinositide phosphatase [Daniorerio] GenBank: BAG50379.1 (SEQ ID NO. 196); TPIP alpha lipidphosphatase [Homo sapiens] GenBank: CAD13144.1 (SEQ ID NO. 197); TPTE2Phosphatidylinositol-3,4,5-triphosphate 3-phosphatase [Homo sapiens]Uniprot: Q6XPS3 (SEQ ID NO: 198)

FIGS. 32A-C show a multiple sequence alignment of the amino acidsequences of human Kv2.1 (SEQ ID NO: 29); CiVSP voltage-sensorcontaining phosphatase [Ciona intestinalis](GenBank: BAD98733.1) (SEQ IDNO. 195); DrVSP voltage-sensing phosphoinositide phosphatase [Daniorerio] GenBank: BAG50379.1 (SEQ ID NO. 196); TPIP alpha lipidphosphatase [Homo sapiens] GenBank: CAD13144.1 (SEQ ID NO. 197); TPTE2Phosphatidylinositol-3,4,5-triphosphate 3-phosphatase [Homo sapiens]Uniprot: Q6XPS3 (residues 1-518 of SEQ ID NO: 198); TPTE (SEQ ID NO:202); and Shaker (SEQ ID NO: 203).

FIGS. 33A-D show the I/V relationships of WT (A), R1S (C). Currents werenormalized to the maximal current at +60 mV (mean data from 7 oocytes(A) and 4 oocytes (C)). Normalized current amplitudes at given voltagesare shown as mean values±S.E. (hidden by symbols). Typical potassiumoutward currents through WT (B) and mutant R1S (D) channels areillustrated on the right panel. The insets display the correspondingvoltage protocols and holding potentials.

FIGS. 34A-D. The I/V curve shows that Iω are not present in WT Shaker(A). Test pulses were applied from +60 mV to −300 mV (illustrated in B).Currents were normalized to the maximal current at +60 mV (data from 7oocytes). Normalized current amplitudes at given voltages are shown asmean values±S.E. (hidden by symbols). Typical potassium outward currentsthrough WT (C) are illustrated on the lower panel. Same experiment athigher resolution is shown in (D) illustrating an unspecific inwardconductance developing at pulses negative to −260 mV (see also meanvalues in A at −280 and −300 mV).

FIGS. 35A-D. The I/V curve shows that Iω is present in R1 S and thatthis current is blocked by La³⁺ (A). Test pulses were applied from +60mV to −300 mV (same voltage steps as in FIG. 34B applied from a holdingpotential of −50 mV). Currents were normalized to the maximal current at+60 mV. Control R1 S I/V curve (squares) and I/V curves in the presenceof 30 (circles), 100 (triangles) and 300 μM La³⁺ (diamonds) are shown(data from 4 oocytes). Normalized current amplitudes at given voltagesare shown as mean values±S.E. Typical potassium outward currents throughR1 S and omega currents in control (B) and in the presence of 30 μM (C)and 300 μM La³⁺ (D) are illustrated.

FIGS. 36A-D. The I/V curve shows that Iω is present in R1S/W434F (A).Test pulses were applied from +60 mV to −300 mV (same voltage protocolas in FIG. 34 B). Current amplitudes at given voltages are shown as meanvalues±S.E (data from 6 oocytes). (B) and (C) illustrate the absence ofoutward currents and at a higher resolution (C) the presence of omegacurrents in construct R1 S/W434F. (D) Iω was induced by voltage stepsfrom a holding potential of −80 mV to −200 mV (see inset). Inhibition ofIω induced by 100 μM La³⁺ occurred in a “use-dependent” manner. Thelower trace (control, in the absence of La³⁺) is superimposed by twentycurrents during 20 ms pulses applied at a frequency of 1 Hz.

FIGS. 37A-H show the I/V relationships of various Kv2.1 channelsconstructs expressed in Xenopus oocytes injected with the correspondingcRNAs: WT (A), R0N (B), R2S (C), R0C/R2S (D), R-1S/R0S (E), R-1S/R0C(F), R-1S/R0S/R2S (G) and R-1S/R0C/R2S (H) Kv2.1 channel constructsexpressed in Xenopus oocytes are illustrated. Currents were normalizedto the maximal current at +60 mV (data from 9 (A), 7 (B), 6 (C), 4 (D),8 (E), 4 (F), 7 (G) and 4 oocytes (H)). Normalized current amplitudes atgiven voltages are shown as mean values±S.E. (hidden by symbols).

FIGS. 38A-H show the typical potassium outward currents though thedesignated channel constructs: WT (A), R0N (B), R2S (C), R0C/R2S (D),R-1S/R0S (E), R-1S/R0C (F), R-1S/R0S/R2S (G) and R-1S/R0C/R2S (H).

FIG. 39 shows the mean maximal outward currents at +60 mV through thedesignated Kv2.1 constructs (in μA; two batches, 3).

FIGS. 40A-F show the I/V relationships for Kv2.1 channels in the absenceand presence of AgTx2. WT (A) and R2S (D) channels are shown in theabsence (control, squares) and presence of 1 nM AgTx2 (circles). Peakcurrents were normalized to the maximal current at +60 mV. Normalizedpeak current amplitudes at given voltages are shown as mean values±S.E.(hidden by symbols). The mean inhibition of the maximal outward currentat +60 mV amounted 66.5±2.9% (n=4, WT) and 67.8±2.8% (n=5, R2S). Typicalpotassium outward currents through WT and R2S Kv2.1 channels in control(B, E) and in the presence of 1 nM AgTx2 (C, F) are illustratedrespectively.

FIG. 41 shows current-voltage relationships of WT, R0N, R2S, R0C/R2S,R-1S/R0C, R-1S/R0S, R-1S/R0C/R2S and R-1S/R0S/R2S Kv2.1 channelconstructs normalized to maximal outward current at +60 mV. Inwardcurrents were recorded during 10 ms hyperpolarising voltage steps from aholding potential of −80 mV to −300 mV (20 mV steps) in Xenopus oocytesinjected with the corresponding cRNAs. Normalized current amplitudes atgiven voltages are shown as mean values±S.E. (n≥4, two batches ofoocytes). Currents were not leak subtracted.

FIG. 42 shows the inward current-voltage relationships of Kv2.1construct R-1S/R0S/R2S in the absence (control, squares) and after 5minutes in the presence of 10 nM AgTx2 (filled circles). Inward currentsafter 5 ms were normalized to the maximal outward current at +60 mV.Normalized inward current amplitudes at given voltages are shown as meanvalues±S.E. (n≥4, two batches of oocytes).

FIGS. 43A-C. (A) illustrates the inward current-voltage relationships ofconstruct R-1S/R0S/R2S in the absence (control; squares) and in thepresence of the indicated concentrations of La³⁺ (other symbols). Inwardcurrents after 5 ms in control and La³⁺ were normalized to the maximaloutward current at +60 mV. Normalized inward current amplitudes at givenvoltages are shown as mean values±S.E. (n≥4, two batches of oocytes).(B, C) illustrate typical inward currents during hyperpolarizing pulsesthrough R-1 S/R0S/R2S in the absence (B) and in the presence of 10 mMLa³⁺ (C).

FIGS. 44A-B. (A) illustrates the concentration-dependent inhibition ofthe inward currents after 5 ms (normalized to control) through R-1S/R0S/R2S at −300 mV. The concentration-inhibition curve was fitted tothe Hill equation. (B) illustrates superimposed inward currents at −300mV in the absence and in the presence of 30 μM, 100 μM, 300 μM, 1 mM, 3mM and 10 mM La³⁺. A half-maximal inhibition concentration (IC50) of785±419 μM was estimated (nH=0.8±0.1; n≥4, two batches of oocytes).

FIG. 45 shows the inward current-voltage relationships of Kv2.1 mutantR-1S/R0C/R2S in the absence of (squares) and after 5 minutes applicationof 1 mM MTSEA (filled circles). Inward currents after 5 ms in theabsence of reagent or in the presence of the MTSEA, MTSES, or MTSETreagents were normalized to the inward current at −300 mV in the absenceof reagent. Normalized inward current amplitudes at the indicatedvoltages are shown as mean values±S.E. (n≥4, two batches of oocytes).

FIG. 46 shows the inward current-voltage relationships of Kv2.1 mutantR-1S/R0C/R2S in the absence of (squares) and after 5 minutes applicationof 1 mM MTSES (filled circles). Inward currents after 5 ms in theabsence of reagent or in the presence of the MTSEA, MTSES, or MTSETreagents were normalized to the inward current at −300 mV in the absenceof reagent. Normalized inward current amplitudes at the indicatedvoltages are shown as mean values±S.E. (n≥4, two batches of oocytes).

FIG. 47 shows the inward current-voltage relationships of Kv2.1 mutantR-1S/R0C/R2S in control (squares) and after 5 minutes application of 1mM MTSET (filled circles). Inward currents after 5 ms in the absence ofreagent or in the presence of the MTSEA, MTSES, or MTSET reagents werenormalized to the inward current at −300 mV in the absence of reagent.Normalized inward current amplitudes at the indicated voltages are shownas mean values±S.E. (n≥4, two batches of oocytes).

FIG. 48 shows alignment of the Shaker (residues 357-386 of SEQ ID NO: 1)and Kv2.1 (residues 292-321 of SEQ ID NO: 29).

FIGS. 49A-B. (A) illustrates the inward current-voltage relationships ofIω of Kv2.1 R-1 S/R0S/R2S in the absence of (squares) and after 5minutes application of 100 nM GxTx-1E (circles). Inward currents after 5ms were normalized to the inward current at −300 mV in the control.Normalized inward current amplitudes at the indicated voltages are givenas mean values±S.E. (B) illustrates typical inward currents duringhyperpolarizing pulses through R-1S/R0S/R2S in the absence of (left) andin the presence of 100 nM GxTx-1E (right).

FIG. 50 shows the inward current-voltage relationships of Iω of Kv2.1R-1S/R0S/R2S in the absence of (squares) and after 5 minutes applicationof 10 mM 2GBI (circles). Inward currents after 5 ms in control and 2GBIwere normalized to the inward current at −300 mV in the control.Normalized inward current amplitudes at the indicated voltages are givenas mean values±S.E.

FIG. 51 shows the inward current-voltage relationships of Iω of Kv2.1R-1S/R0S/R2S in the absence of (squares) and after 5 minutes applicationof 10 μM B1 (circles). Inward currents after 5 ms in control and B1 werenormalized to the inward current at −300 mV in the control. Normalizedinward current amplitudes at the indicated voltages are given as meanvalues±S.E.

FIG. 52 shows the inward current-voltage relationships of Iω of Kv2.1R-1S/R0S/R2S in the absence of (squares) and after 5 minutes applicationof 10 μM RY785 (circles). Inward currents after 5 ms in control andRY785 were normalized to the inward current at −300 mV in the control.Normalized inward current amplitudes at the indicated voltages are givenas mean values±S.E.

FIGS. 53A-C show a multiple sequence alignment of the amino acidsequences of Drosophila Shaker (SEQ ID NO:1), human Kv2.1 (SEQ ID NO:29); CiVSP voltage-sensor containing phosphatase [Cionaintestinalis](GenBank: BAD98733.1) (SEQ ID NO. 195); DrVSPvoltage-sensing phosphoinositide phosphatase [Danio rerio] GenBank:BAG50379.1 (SEQ ID NO. 196); TPIP alpha lipid phosphatase [Homo sapiens]GenBank: CAD13144.1 (SEQ ID NO. 197); TPTE2Phosphatidylinositol-3,4,5-triphosphate 3-phosphatase [Homo sapiens]Uniprot: Q6XPS3 (SEQ ID NO: 198)

DETAILED DESCRIPTION OF THE INVENTION

The issued patents, applications, and other publications that are citedherein are hereby incorporated by reference to the same extent as ifeach was specifically and individually indicated to be incorporated byreference.

Described herein are methods for identifying compounds that interactwith VGPs and modulate their ionic conductance or enzymatic activity orother functional property, and/or change the distribution of theirfunctional and/or conformational states. In certain aspects, theinvention relates to methods for identifying compounds capable ofmodulating the activity of one or more types of voltage-gated proteins.The methods described herein can be use alone or in conjunction with anyother screening methods known in the art and can be used in connectionwith other methods known in the art to identify compounds, mutations,biological mechanisms or therapeutic treatments, including, but notlimited to those methods that employ combinatorial chemistry, molecularbiology, high throughput screening, structure-based drug design, invitro, in-vivo, in-silico methods, and the like.

Definitions

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise.

The term “about” is used herein to mean approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 20%.

As used herein, the term Voltage-Gated Protein (VGP) refers to a proteincomprising a domain capable of sensing a voltage (e.g., a voltagesensing domain). Many such VGPs are known in the art and can be used inconnection with the methods described herein. Examples of VGPs suitablefor use with the methods described herein include, but are not limitedto proteins that form ion channels, for example, voltage-gated ionchannel (VGIC) proteins, as well proteins that do not form ion channels,for example voltage sensing phosphatase proteins (VSP).

Examples of VSPs suitable for used with the methods described hereininclude, but are not limited to CiVSPs, and TPTE proteins. VGICs are asuperfamily comprising at least four major families of ion channels,each of which have functionally relevant sequence similarity as well asa varying degree of functional similarity. VGICs are also classified bythe type of ions that pass through the channels, including potassiumchannels, calcium channels, sodium channels, and proton channels. Othertypes of channels include protein channels, chloride channels, and waterchannels (aquaporins). Thus, as used herein the term VGIC can refer to avoltage-gated potassium channel (VGPC), a voltage-gated calcium channel(VGCC), a voltage-gated sodium channel (VGSC), a voltage-gated protonchannel (VGHC), or a voltage activated phosphatase (CiVSP). In certainembodiments, the term VGP, VGIC or VSP refers to a human VGP, VGIC orVSP protein however the use of the terms VGP, VGIC or VSP is notintended to be limited and encompass non-human orthologues andhomologues found in other species, including insect and worm species.Where insect or worm VGPs are used in connection with the methodsdescribed herein, compounds identified using the methods describedherein can, in certain embodiments, be useful as pesticides or toxins.VGPs from non-human sources suitable for use with the methods alsoinclude, but are not limited to, UniProtKB/Swiss-Prot: Q4W8A1; F6XHE4;F6XHF1; E1BUX1; F6LWC2; F1QG29; Q4V9E4; B3IUN7; G1NQ81; A8WGV0; A8WGV0;Q4KLP3; G3QBH0; Q4SFW2; F7DBU7; F6X178; G3TRR6; G1KSZ1; F7DBQ4; F1P987;G1LZ63; Q3KNE1; G5E8H5; E9Q3G1; G1TEB2; Q91X03; Q91X02, or variants,homologues or orthologues thereof.

One of skill in the art will appreciate that because VGPs areevolutionarily conserved among species, functionally related homologuesof human VGPs exist in other species. A functional relationship betweenhomologous proteins can be indicated in a number of ways, including, butnot limited to: (a) the degree of sequence identity; and/or (b) the sameor similar biological function. Homology can be determined usingsoftware programs readily available in the art, such as those discussedin Current Protocols in Molecular Biology (F. M. Ausubel et al., eds.,1987). Methods for identifying sequence identity between proteins andfunctional identity between amino acids between two or more VGPsequences are well known in the art, and include BLAST alignment as wellas other methods described herein. One of skill in the art will alsounderstood that substitutions of amino acids at functionally similarpositions between different ion channels will result in a mutant ionchannel capable of forming a transmembrane pore, alone or in thepresence of one of more ion channel proteins. Accordingly, reference toa specific amino acid position in a human VGP will also refer to theamino acid occupying the same position in a related VGP sequence fromanother species. Because one of skill in the art will be capable ofperforming multiple sequence alignment using readily available tools,the skilled artisan will be capable of aligning the amino acid residuesof the VGPs shown in FIG. 15 against VGPs, VGICs or VSPs from non-humanspecies, including VGPs from insects and worms. For example, unlessspecifically indicated otherwise, it will be understood that thethreonine residue at position 286 of the human Kv1.1 polypeptidesequence is functionally related to the methionine residue at position356 of the Drosophila Shaker polypeptide sequence. One of skill in theart will also appreciate that because splice variants of RNA transcriptsencoding VGICs are know in the art, reference to a specific amino acidposition in a VGIC will also refer to the amino acid occupying the sameposition in a VGIC encoded by a splice variant RNA or that whichcorresponds to a splice variant isoform of a VGIC. One of skill in theart will also appreciate that VGICs from other species can be used inconnection with the methods described herein. Accordingly, one of skillin the art will readily be capable of introducing amino acidsubstitutions in non-human VGICs by aligning the sequence of thenon-human VGIC with the sequences of the VGICs described herein.

As used herein, the activity of a VGP can refer to the activity of aVGIC or the activity of any other protein that is regulated by voltage,for example a VSP. One of skill in the art will appreciate that theactivity of a VGP can also be modulated by compounds that bindirreversibly or reversibly to the VGP, or compounds that bindirreversibly or reversibly to another protein or molecule that regulatesthe activity of the VGP. Where a compound regulates the activity of aVGP, one of skill in the art will readily be capable of determining theamount of the compound required to modulate (e.g. increase or decrease)the activity of the VGP, for example by determining the IC50 of thecompound as it relates to its ability to modulate the activity of theVGP.

Where the VGP is a VSP, reference to the activity of the VSP can referto the rate or ability of the VSP to dephosphorylate a target. Forexample, Ci-VSP dephosphorylates phosphatidylinositol 3,4,5-bisphosphate(PIP3) to phosphatidylinositol 4,5-bisphosphate (PIP2) upon membranehyperpolarization. The methods described herein can comprise a step ofintroducing one or more mutations in a VSP that cause the VSP to exhibitaltered voltage sensitivity. In certain embodiments, the mutation(s)causing the VSP to exhibit altered voltage sensitivity can be a mutationin one or more VSD(s) of the VSP. In certain embodiments, themutation(s) can be in VSD transmembrane helices. In certain embodiments,the mutation(s) in the VSD transmembrane helices are mutations of aminoacids facing the center of the VSD. In certain embodiments, themutations can be located on VSD helix S4. In certain embodiments, themutations are to one or more conserved arginine or lysine residues inhelix S4. In certain other embodiments, the mutations are to one or moreconserved aspartate or glutamate residues located elsewhere in the VSD.The mutated VSD will exhibit, by virtue of these mutations, abnormalvoltage sensitivity of the VSD; by contrast, the non-mutated (“wildtype”) VSD exhibits no such altered voltage sensitivity. In certainaspects, the methods described herein relate to screening assayssuitable for monitoring altered voltage sensitivity of a mutated VSP.

Where the VGP is a VGIC, reference to the activity of the VSP can referto the rate or absolute amount of ion permeation through an ion channel.In certain embodiments, the permeation can be a measure of ionpermeation through the alpha pore of the channel. In certainembodiments, VGIC activity as measured as a function of ion permeation,can be a measure of ion permeation that occurs independently from alphapore permeation, for example permeation through an omega leak or a sigmaleak. In certain embodiments, the activity of a VGIC can refer to theselectivity of an ion channel. For example, in certain embodiments, themethods described herein can be useful of determining whether a compoundalters permeability of a channel for one ion as compared to another ion(e.g. calcium or potassium). A modulation of the activity of a VGIC canbe reflected in a change in the VGIC activity as a function of voltage.For example, in certain embodiments, the activity of a VGIC will referto the voltage of half-maximal ion permeation. In other embodiments, theactivity of a VGIC can refer to the sensitivity of voltage dependentpermeation. Methods for measuring the sensitivity of ion permeation areknown in the art, and include, for example determining current-voltage(I-V) and conductance-voltage (G-V) curves, and/or including fits to theBoltzmann equation. The activity a VGIC can also refer to regulation ofion permeation through the VGIC that occurs as a result ofpost-translational modifications, for example by proteinphosphorylation. In certain embodiments, the methods of the inventionare useful for distinguishing between VGIC binding molecules that (a)that bind preferentially to a VGIC when the VGIC is in an open oractivated functional state, (b) that bind preferentially to a VGIC in aclosed or inactivated functional state, or (c) that bindnon-preferentially to a VGIC in either a closed or inactivated, or openor activated, functional state. “Open” or “activated” both refer to afunctional state of the VGIC that is competent to conduct ions, whereas“closed” or “inactivated” both refer to functional states of the VGICthat is not competent to conduct ions. For those VGICs that are not ionchannels, the terms “open” and “closed” lose meaning; accordingly, forthese proteins “activated” and inactivated” refer to functional statesof the VGIC that are competent, or not, with respect to a particularfunctional property of the protein (e.g., an enzymatic activity),respectively. The methods described herein are suitable for identifyingany number of compounds that bind VGICs, including, but not limited tosmall molecules, peptides, proteins, and antibodies, or derivatives andfragments thereof.

Voltage-Gated Protein Activity

Changes in the conformational configuration of VGPs occur in response tochanges in membrane voltage. Voltage sensitivity of VGPs is provided byvoltage sensor domain comprising S1, S2, S3 and S4 segments of VGPs. Thevoltage sensing function of the voltage sensor domain depends primarilyon the presence of positively charged lysine or arginine residues (i.e.gating charges) within a transmembrane helical segment called the S4segment. The S4 segment of VGPs typically contains between 4 to 8 gatingcharges capable of moving in response to changes in membrane potential.Movement of these positively charged residues in the S4 segment in theelectric field of the membrane results in changes to the position of S6segments and thereby triggers graded conformational changes of the VGPbetween closed (e.g., inactivated) and open (e.g. activated) states uponchanges in membrane potential. Many VGPs exhibit significant voltagedependence upon changes in membrane potential wherein activity increases10-fold for every 7 to 12 mV of changes in membrane potential.

In certain embodiments, determining VGP activity according to themethods described herein can comprise a step of stimulating a membranecomprising a VGP. Examples of stimuli suitable for use with the methodsdescribed herein include, but are not limited to, electricalstimulation, magnetic stimulation, chemical stimulation, biologicalstimulation, or combinations thereof. Where chemical stimulation isused, introduction of the stimulus can comprise contacting an ionchannel comprised in a membrane with a salt (e.g. a sodium salt or apotassium salt). Chemical stimulation can also comprise contacting amembrane comprising a VGP with any pore forming molecule known in theart. Where biological stimulation is used, the stimulus can comprise astep of contacting a membrane comprising a VGP with a chemical orbiological compound that modifies the activity of the VGP. Wheremagnetic stimulation is used, the stimulus can comprise exposing an ionchannel comprised in a membrane to an alternating magnetic field. Whereelectrical stimulation is used, the introduction of the stimulus cancomprise the use of a patch clamp and the application of an externalelectric field to an ion channel comprising a membrane.

One of skill in the art will readily be capable of determining the type,amplitude, intensity, concentration or frequency of a stimulus suitablefor use with the methods described herein. For example, in the case ofelectrical stimulation, the voltage amplitude and the duration ofstimulation can be selected on the basis of the activation kinetics ofthe VGP being examined. For example, in certain embodiments, an ionchannel comprised in a membrane can be maintained at a first membranepotential prior to being subjected to a depolarizing pulse. The membranecomprising the VGP can then be returned to the first membrane potential.In certain embodiments, the membrane comprising the VGP can be subjectedto a second membrane potential. In one embodiment, the second membranepotential will be more positive that the first membrane potential. Inanother embodiment, the first membrane potential will be more positivethat the second membrane potential. Specific examples of first membranepotential voltages and second membrane potential voltages can beindependently selected from about −300 mV, about −280 mV, about −260 mV,about −240 mV, about −220 mV, about −200 mV, about −180 mV, about −160mV, about −140 mV, about −120 mV, about −100 mV, about −80 mV, about −60mV, about −40 mV, about −20 mV, about 0 mV, about 20 mV, about 40 mV,about 60 mV, about 80 mV, about 100 mV, about 120 mV, about 140 mV,about 160 mV, about 180 mV, about 200 mV, about 220 mV, about 240 mV,about 260 mV, about 280 mV, about 300 mV, and ranges between any two ofthese values.

In certain embodiments, the VGP monitoring methods described herein cancomprise a step of exposing the membrane comprising the VGP to at leastone step voltage prior to subjecting them to the depolarizing pulse at asecond membrane potential voltage. A step voltage can be a voltage thatis between the voltage of the first membrane potential and the secondmembrane potential. For VGICs, in certain embodiments of the methodsdescribed herein, the step voltage can be used to measure non-specificleak currents that need to be subtracted to obtain the specific leakcurrent of interest. For example, a monitoring method described hereincan comprise a first membrane potential voltage of about −70 mV, a stepvoltage of about −40 mV, and a second membrane potential voltage ofabout 20 mV.

In embodiments where a depolarizing pulse is used in connection with themethods described herein, the depolarizing pulse can be applied for anylength of time suitable for monitoring the activity of an ion channel.Suitable depolarizing pulse time lengths include about 10 microseconds,about 1 milliseconds, about 10 milliseconds, about 100 milliseconds,about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about5 seconds, about 10 seconds, about 20 seconds, about 30 seconds, about40 seconds, about 50 seconds, about 60 seconds, about 70 seconds, about80 seconds, about 90 seconds, about 100 seconds, about 500 seconds,about 1000 seconds, about 2000 seconds, about 3000 seconds, about 4000seconds, about 5000 seconds, and ranges between any two of these values.

Membrane depolarization can also be performed by altering ionconcentrations. For example, in certain embodiments of the methodsdescribed herein, membrane depolarization can be performed by theaddition of positive ions (e.g. K+ ions) into a medium comprising a VGPin a membrane (e.g. into a solution comprising a cell expressing theVGP) to induce depolarization by shifting the equilibrium potential. Incertain embodiments, the amount of a positive ion added to the mediumcan be an amount sufficient to shift the equilibrium potential to thepositive direction by about 30 mV, about 35 mV, about 40 mV, about 45mV, about 50 mV, about 55 mV, about 60 mV, about 65 mV, about 70 mV,about 75 mV, about 80 mV, or more than about 85 mV. One of skill in theart will appreciate that the amount of depolarization will depend on theamount of ion added and that a desired amount of depolarization candepend on the VGP selected for investigation in conjunction with themethods described herein.

Other methods for depolarizing a membrane (e.g. the membrane of a cell)are also suitable for use with the methods described herein. Forexample, in certain embodiments, membrane depolarization can beperformed with the use of potassium channel blockers, ion channelmodulating agents. Examples of potassium channel blockers suitable foruse with the methods described herein include, but are not limited toCs⁺, Ba²⁺, and tetraethylammonium (TEA). Examples of small organicmolecules suitable for use with the methods described herein include,but are not limited to, 4-aminopyridine, quinidine or phencyclidine.Examples of toxins suitable for use with the methods described hereininclude, but are not limited to, charybdotoxin, margatoxin, iberiotoxin,noxiustoxin, kaliotoxin.

Measuring Currents of VGICs

In certain aspects, the methods described herein relate to the use ofion channels that exhibit independent ion permeation. As used herein,the term “independent ion permeation” includes omega pore leak currents(i.e., omega pore dependent ion permeation) and sigma pore leak currents(e.g. sigma pore dependent ion permeation). Independent ion permeation(e.g., ion permeation that occurs elsewhere from the alpha pore) refersto ion permeation that occurs at a resting state potential as well asion permeation that occurs at a non-resting state potential. Independention permeation also refers to ion permeation that occurs under polarizedconditions and non-polarized conditions. As used herein, the term“polarized” refers to the existence of a voltage difference across astructure (e.g., a lipid bilayer, a membrane or a supporting layer)comprising a VGIC, a VGIC variant, or a fragment of a VGIC or a VGICvariant. In certain embodiments, the voltage difference across astructure can arise from a difference in the amount or concentration ofcharged species at one side of the structure relative to the other sideof the structure. In certain embodiments, the voltage difference acrossa structure can arise from an applied voltage. In certain embodiments,the voltage difference across a structure can arise from a difference inthe amount or concentration of charged species at one side of thestructure relative to the other side of the structure in combinationwith an applied voltage.

The methods described herein can be performed by modifying an ionchannel to induce the channel to exhibit non-alpha-pore ion conductionwhen the channel is in any of an open, closed, activated, or inactivatedstate. In certain respects, the methods described herein can be appliedby introducing one or more mutations in a VGIC analogous to knownmutations that cause VGICs to exhibit alpha-pore-independent ionconduction. The compounds identified by the methods described hereinwill be useful for the treatment of voltage-gated ion channel relatedchannelopathies and other pathological or physiological conditionsrelated to abnormal voltage-gated protein function.

VGICs that exhibit alpha-pore-independent ion conduction can be screenedaccording to any method known in the art. For example, in certainembodiments, a VGIC that exhibits alpha-pore-independent ion conductioncan be screened by expressing the VGIC in a cell and testing theactivity of the VGIC both in the presence and absence of a testcompound. Alternatively, the VGICs described herein can also be testedby expressing the VGIC in a cell prior to isolating the expressed VGICand inserting the VGIC in an artificial membrane (e.g. a liposome)wherein the extracellular portion of the VGIC is on the first side ofthe membrane, and the intracellular portion of the VGIC on the secondside of the membrane such that the VGIC forms a pore between a firstside of the membrane and the second side of the membrane. In certainembodiments, the membrane can be impermeable to a test compound.

VGIC activity is reflected as a change in, for example, ion conductivityor catalytic activity, which depends upon changes in the potentialacross a membrane (e.g. a cell membrane). Membrane potential is thevoltage difference between the inside and the outside of a membrane.Methods of measuring ion conduction or catalytic activity in accordancewith the methods described herein can be by any method known in the art.Activity of a VGIC can be measured directly or indirectly. Directmethods include, but are not limited to, measuring, for a VGIC, a changein the concentration of one or more types of ions at one or both sidesof a membrane; direct electrical measurement of the ionic currentflowing across the membrane, or product formation or substrateconsumption for catalytic activity of a VGIC. Indirect measurements cancomprise measuring changes in membrane potential or changes in pH. Alsosuitable for use with the methods described herein is the use offunctional metrics to measure the downstream effects of VGIC function.For example, when the VGICs described herein are expressed in an intactcell, cell specific effects can be determined as indicator of ionconduction through the VGIC. Examples of downstream effects of VGICfunction include, but are not limited to, transmitter release (e.g.,dopamine), hormone release (e.g., insulin), transcriptional changes togenetic markers (e.g., northern blots), cell volume changes (e.g., inred blood cells), immunoresponses (e.g., T cell activation), changes incell metabolism such as cell growth or pH changes, and changes inintracellular second messengers such as [Ca2+].

In certain embodiments, the methods described herein involve a step ofmeasuring membrane potential with patch clamp techniques (e.g. using amicroelectrode, which is a saline-filled glass micropipette that impalesa membrane comprising a VGIC) or through the use of voltage sensitiveoptical probes. Examples of optical probes suitable for use with themethods described herein include, but are not limited to those describedin Gonzalez and Tsien, 1997.

The ability of a test compound to alter the permeations of a VGICdescribed herein can be determined by assessing a change in electricalpotential of a cell or a membrane comprising the VGICs described herein.In certain embodiments, the changes in cellular polarization in thepresence or absence of a test compound can be performed by measuringwhether there is a change in current through a VGIC when the VGIC iscontact with a test compound. The concentration of the test compoundsused in conjunction with the methods described herein can be anyconcentration that affects alpha-pore-independent ion conduction of theVGICs described herein. In certain embodiments, the concentration of atest compound is in the range of 1 pM to 100 mM.

Any method known in the art for measuring the activity of a VGIC can beused in connection with the methods described herein and include but arenot limited to those described in Gonzalez et al., Drug Discov Today.1999 September; 4(9):431-439. Exemplary methods include, but are notlimited to techniques that employ patch clamp measurement, fluorescencemeasurement, absorbance measurement, radio labeled measurement, andbiological assay measurement (e.g., Ca2+ release, hormone, pH, etc.).

In certain embodiments, the methods described herein can includemeasuring conductance through a VGIC using a patch clamp technique. Inpatch clamp techniques, a voltage clamp can be used to control themembrane potential across a membrane. The circuit resistance in a patchclamp measurement technique can depend on the characteristics of theassay system. For example, resistance in an oocyte system can bedifferent than resistance in a cell line system. The aperture of thepipette used in a patch clamp procedure can be any suitable size (e.g.about 1 μm), however, one of skill in the art will readily be capablefor determining an appropriate membrane patch size for a particularassay condition. Generally, the patch clamp procedure involves placementof the polished aperture of a glass pipette against a membranecomprising the test VGIC. The application of suction results in theformation of a resistant seal between the membrane and the pipette. Thecurrent measured in a patch clamp technique will be the current passingthrough the VGICs and the leak currents escaping between the pipette andthe membrane, through the membrane, and through other ion channels. Oneof skill in the art will readily be capable of determining theappropriate degree of sealing required between the pipette and themembrane suitable for obtaining patch clamp recordings.

Several different patch clamp methodologies are known in the art and aresuitable for use with the methods described herein. Such patch clampmethods include, but are not limited to whole cell perforated patchclamp methods, cell attached patch clamp methods, conventional wholecell methods, and excised (inside out) patch clamp methods. Alsosuitable for use with the methods described herein are planar patchclamp methods. For planar patch clamping, a membrane (e.g. a cellmembrane) comprising a VGIC is attached by suction to an aperture in aplanar substrate. This step eliminates the need for manual manipulationof a glass pipette as otherwise required for certain traditional patchclamp techniques. Patch clamp methods can be used to record ion channelactivity in sub-millisecond time scales. In addition to the study ofpopulations of ion channels expressed in a cell, methods are known inthe art to adapt patch clamp methods for studying the activity of singleion channels.

The methods described herein can be performed by using any patch clampmethod known in the art that is suitable for analyzing ion channelfunction (Neher, E. and Sakmann, B., Nature 260(5554): 799-802 (1976);Hamill, O. P., et al., Pflugers Arch. 391(2): 85-100 (1981)). In certainembodiments, patch clamp analysis can be performed by contacting a cellexpressing a VGIC with the tip of a glass micropipette to obtain aleakage resistant seal (>1 GOhm, GigaSeal). Generally, in the whole-cellconfiguration of the patch clamp method, the intra-pipette portion ofthe membrane makes direct electrical contact between the cell interiorand the pipette electrode. The method allows the application ofdifferent voltages to the pipette electrode whereby the measuredcurrents represent the current through the cell membrane. This currentthrough the cell membrane include current passing through ion channelsexpressed in cellular membranes. In one embodiment, the methodsdescribed herein can comprise a step of monitoring the activity of anion channel using the two electrode voltage clamp technique. In the twoelectrode voltage clamp technique, two electrodes are used wherein oneelectrode is dedicated to the passing current whereas a second electrodeis dedicated to voltage recording. Where two electrodes are used tomonitor ion channel activity, it can be desirable to express an ionchannel selected for monitoring in a cell that is of sufficient size tofacilitate the use of two electrodes. Accordingly, because oocytes ofthe African clawed toad (Xenopus laevis) are large in size, the twoelectrode method can be performed on Xenopus oocytes that have beenmicroinjected with cDNA or cRNA encoding an ion channel selected formonitoring. Other patch clamp methods suitable for use with the methodsdescribed herein are also known, and include, but are not limited to the“cell-attached” patch clamp technique, the “inside-out” patch clamptechnique, and the “whole cell” patch clamp technique (see, e.g.,Ackerman et al., New Engl. J. Med. 336:1575-1595 (1997); Hamil et al.,Pflugers. Archiv. 391:85 (1981)).

In certain embodiments, florescent probes and dyes can be used to detectthe intracellular or subcellular concentrations of ions. One of skill inthe art will appreciate that several different dyes are available withdifferent affinity ranges and applications.

Voltage sensitive dyes responsive to changes in membrane potential canbe used to measure absolute membrane potentials as well as to measurechanges in membrane potential in accordance with the methods describedherein. Such changes in membrane potential can be use as a non-linearreadout of a change in ion channel activity. The responsiveness ofvoltage sensitive dyes to changes in membrane potential occur as aresult of changes in the distribution of the intramolecular charges,which then results in a change in their fluorescent emission intensityand spectral patterns. In certain embodiments, the dye used to measuremembrane potential can be the fluorescent dye bis-(1,3-dibutylbarbituricacid) trimethine oxonol.

Any voltage sensitive dye suitable for detecting a change in membranepotential can be used in conjunction with the methods described herein,including, but not limited to, coumarin dyes, anionic and hybrid oxonoldyes, hemicyanine dyes, merocyanine dyes, cationic or zwitterionicstyryl dyes, and cationic carbocyanines and rhodamines. Examples ofcoumarin dyes suitable for use with the methods described hereininclude, but are not limited to,N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanol-amine(CC2-DMPE). Examples of anionic and hybrid oxonol dyes suitable for usewith the methods described herein include, but are not limited to,bis-oxonol, oxonol V (bis-(3-phenyl-5-oxoisoxazol-4-yl)pentamethineoxonol), oxonol VI (bis-(3-propyl-5-oxoisoxazol-4-yl)pentamethineoxonol), bis-(1,3-diethylthiobarbituric acid)trimethine oxonol(DiSBAC.sub.2(3), bis-(1,3-dibutylbarbituric acid)trimethine oxonol(DiBAC.sub.4(3), bis-(1,3-dibutylbarbituric acid)pentamethine oxonol(DiBAC.sub.4(5), RH-155 (NK3041), RH-479 (JPW1131), RH-482 (JPW1132,NK3630), RH-1691, RH-1692, RH-1838, RH-1114 (WW781), JPW1177, andJPW1245. Examples of hemicyanine dyes suitable for use with the methodsdescribed herein include, but are not limited to,dibutylamino-naphthalene-butylsulfonato-isoquinolinium (BNBIQ). Examplesof merocyanine dyes suitable for use with the methods described hereininclude, but are not limited to, merocyanine 540, NK2495 (WW375), andJPW1124. Examples of cationic or zwitterionic styryl dyes suitable foruse with the methods described herein include, but are not limited to,di-4-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate(di-4-ANEPPS),di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate(di-8-ANEPPS), di-12-ANEPPS, di-18:2-ANEPPS, di-2-ANEPEQ (JPW1114),di-12-ANEPEQ, di-8-ANEPPQ, di-12-ANEPPQ, di-1-ANEPIA, D-6923 (JPW3028),N-(4-sulfobutyl)-4-(6-(4-(dibutylamino)phenyl)hexatrienyl) pyridinium(RH-237),N-(3-triethylammoniumpropyl)-4-(4-(4-(diethylamino)phenyl)butadienyl)pyridiniumdibromide (RH-414),N-(4-sulfobutyl)-4-(4-(4-(dipentylamino)phenyl)butadienyl) pyridinium(RH-421), RH-437, RH-461, RH-795, JPW1063, and FM1-43. Examples ofcationic carbocyanines and rhodamines suitable for use with the methodsdescribed herein include, but are not limited to,3,3′-diethyloxacarbocyanine iodide (DiOC.sub.2(3)),3,3′-dihexyloxacarbocyanine iodide (DiOC.sub.6(3)),3,3′-dimethyl-naphthoxacarbocyanine iodide (JC-9; DiNOCl(3)),3,3′-dipentyloxacarbocyanine iodide (DiOC.sub.6(3)),3,3′-dipropylthiadicarbocyanine iodide (DiSC.sub.3(5)),1,1′,3,3,3′,3′-hexamethylindodicarbocyanine iodide (DilC.sub.1(5)),rhodamine, rhodamine 123,5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanineiodide (CBIC.sub.2(3)), tetramethylrhodamine, ethyl ester, perchlorate(TMRE), and tetramethylrhodamine, methyl ester, perchlorate (TMRM).Other voltage sensitive dyes suitable for use with the methods describedherein include those described in Grinvald et al., 68(4) Physiol. Rev.1285-1366 (1988); Lowe and Goldfinch, 137 Methods Enzymol. 338-348(1988); Katerinopoulos, 10(30) Curr. Pharm. Des. 3835-3852 (2004);Johnson, Fluorescent Probes for Living Cells 30(3) Histochem. J. 123-140(1998); IMAGING NEURONS: A LABORATORY MANUAL (Rafael Yuste, et al.,eds., Cold Spring Harbor Laboratory Press, 2000).

In certain embodiments, the methods described herein are performed usingfast response dyes, slow response dyes, or a combination of both fastand slow response dyes. Fast response dyes are dyes that undergo rapidintramolecular charge distribution upon electrical field changes whichalter the spectral properties and/or the fluorescence intensity of thedye. In general, fast response dyes can be used to detect millisecondtime scale changes in membrane potential. In certain embodiments, fastresponse dyes can be used in connection with the methods describedherein where the method comprises monitoring the conductance of a VGICin an excitable cell, such as a neuron or a cardiac cell. Examples offast response dyes suitable for use with the methods described hereininclude, but are not limited to, di-2-ANEPEQ (JPW1114), di-1-ANEPIA,di-8-ANEPPQ, di-12-ANEPPQ, di-4-ANEPPS, di-8-ANEPPS, di-18:2-ANEPPS,RGA-30, RH-155, RH-795, RH-237, RH-421, RH-414, and WW 781. Slowresponse dyes are generally lipophilic anions or cations that can beused to detect changes in membrane potential as a function of theirtransmembrane distribution. For example, DiBAC dyes have a slowerresponse time as compared to di-ANEPPS dyes because they generally needto traverse a lipid membrane and bind to an intracellular component inorder to produce a fluorescent signal. In certain embodiments, slowresponse dyes can be used in connection with the methods describedherein where the method comprises monitoring the conductance of a VGICin a non-excitable cell as a result of respiratory activity, ion-channelpermeability, drug binding, or other factors. Examples of slow responsedyes suitable for use with the methods described herein include, but arenot limited to, DiSBAC.sub.4(3), DiBAC.sub.4(5), DiBAC.sub.4(3),DiOC.sub.5(3), DiOC.sub.6(3), DiSC.sub.3(5), DiOC.sub.2(3),DiNOC.sub.6(3), DiIC.sub.2(5), merocyanine 540, Oxonol V, Oxonol VI,rhodamine 123, TMRM, TMRE, and CBIC.sub.2(3).

In certain embodiments, the methods described herein can be used inconjunction with calcium sensitive dyes. For example, using fluorescentprobes such as Fluo-3 and Calcium Green can be used to monitor cellular,and subcellular, calcium concentrations as a function of ion channelactivity. In general, free calcium concentration in a cell is about 100nM, however one of skill in the art will readily be capable of measuringthe concentration of free calcium in a cell.

Ion channel activity can also be monitored by measuring the transport ofions across a membrane. For example, in the case of many potassiumchannels, rubidium ions can be used as a tracer and fluorescent dyesthat are sensitive to rubidium can be used to determine membranepermeability to rubidium (Terstappen, 1999; Vestergarrd-Bogind et al., JMembrane Biol. 88:67-75 (1988); Daniel et al., J. Pharmacol. Meth. 25:185-193 (1991): Holevinsky et al. J. Membrane Biology 137:59-70 (1994)).

Ion channel activity can also be monitored by measuring the transport ofions across a membrane. For example, in the case of many potassiumchannels, thallium ions (Tl⁺) can be used as a tracer and fluorescentdyes that are sensitive to thallium can be used to determine membranepermeability to thallium (e.g., Weaver et al. (2004) “AThallium-Sensitive, Fluorescence-Based Assay for Detecting andCharacterizing Potassium Channel Modulators in Mammalian Cells” Journalof Biomolecular Screening 9(8):671-677; Wang et al. (2011) Selectiveinhibition of the K(ir)2 family of inward rectifier potassium channelsby a small molecule probe: the discovery, SAR, and pharmacologicalcharacterization of ML133. ACS Chem. Biol. 6(8):845-856).

Also suitable for use with the methods described herein arevoltage-sensing Fluorescence Resonance Energy Transfer (FRET) acceptors(Gonzalez and Tsien, 4(4) Chem. Biol. 269-277 (1997); U.S. Pat. Nos.5,661,035; 6,107,066). FRET can be used to measure changes in membranepotential in accordance with the methods described herein by determiningthe amount of energy transfer and quenching of fluorescence intensitybetween a donor fluorophore and an acceptor fluorophore. In certainembodiments, membrane depolarization can be detected as a function of achange in donor fluorescence, acceptor fluorescence, or in the emissionmaxima of a donor fluorophore and/or acceptor fluorophore. Examples ofdonor fluorophore and acceptor fluorophore pairs suitable for use withthe methods described herein include, but are not limited to, CC2-DMPEand DiSBAC²(3); CC2-DMPE and DiSBAC⁴(3); CC2-DMPE and RH-155 (NK3041);CC2-DMPE and RH-479 (JPW1131); CC2-DMPE and RH-482 (JPW1132, NK3630);CC2-DMPE and RH-1691; CC2-DMPE and RH-1692; CC2-DMPE and RH-1838;CC2-DMPE and R-1114 (WW781); CC2-DMPE and JPW1177; and CC2-DMPE andJPW1245.

Suitable voltage-sensing FRET acceptors include, but are not limited to,coumarin-tagged phospholipids integrated into the cell membrane. Forexample, coumarin-phospholipid CC2-DMPE partitions to the extracellularhalf of the plasma membrane and is excited by 405 nm light and can beused in connection with the methods described herein.

In embodiments where florescence or light emission is detected as ameasure of ion channel conductance (e.g. where voltage-sensitive dyesare used), the emission of fluorescence or light can be detected by anysuitable method known in the art. For example, in certain embodiments,the emission of fluorescence or light can be detected with the use of afluorimeter.

In certain embodiments, fluorescence or light emission based methods formeasuring ion channel function can be performed using high-throughputformats including, without limitation, 96-well, 384-well or 1536-wellplates. Instruments useful for high-throughput measurement offluorescence or light emission include, but are not limited toFluorometric Imaging Plate Reader or a Voltage/Ion Probe Reader (U.S.Pat. No. 6,342,379; Gonzalez and Maher, 8(5-6) Receptors Channels283-295, (2002); U.S. Pat. No. 6,686,193).

Where fluorescence or light emission is used as a measure of ion channelconductance, a change in ion channel function can be detected as achange (e.g. an increase or decrease) in fluorescence or light emissionrelative to a control sample. In certain embodiments, the change will begreater than about a 5% change in fluorescence or light emission,greater than about a 10% change in fluorescence or light emission,greater than about a 20% change in fluorescence or light emission,greater than about a 30% change in fluorescence or light emission,greater than about a 40% change in fluorescence or light emission,greater than about a 50% change in fluorescence or light emission,greater than about a 60% change in fluorescence or light emission,greater than about a 70% change in fluorescence or light emission,greater than about a 80% change in fluorescence or light emission,greater than about a 90% change in fluorescence or light emission, orgreater than about a 100% change in fluorescence or light emission.

In certain embodiments, the methods described herein can includemeasuring conductance through a VGIC using a radiolabel measurementtechnique. Many radiolabelling methods are known in the art. Forexample, ⁸⁶Rb+ can be used as a flux tracer because it can permeatethrough VGPC as well as other channels having similar properties,however radioactive tracers for Na+, Ca2+ and Cl− also are available(see generally, Terstappen G C. (2004) Assay Drug Dev. Technol. 2:553-559). Generally, the radioactive tracer is incubated with the ionchannel (either in a cell or on a support, such as a membrane) and theexcess tracer is then removed to determine channel activity. In certainembodiments, the channels can be retained in an open or a closedconformation and additional compounds or agents can be contacted withthe channel to determine whether the compound or the agent modifiespermeation of the radioactive tracer through the channel. One of skillin the art will readily be capable of measuring either influx or effluxusing radioactive tracers.

In certain embodiments, the methods described herein can includemeasuring VGIC activity with a biological assay. For example, theactivity of the VGICs described herein can be measured, as non-limitingexamples, by the ability to be regulated by ligands, including knownneurotransmitters, agonists and antagonists, including but not limitedto serotonin, acetylcholine, nicotine, and GABA. Alternatively, theactivity of the ion channel can be assayed by examining their ability tomodulate intracellular Ca2+ concentration, a mitogenesis assay, a MAPKinase activity assay, an arachidonic acid release assay (e.g., using[3H]-arachidonic acid), induce gene transcription or expression,modulate extracellular acidification rates, as well as other binding orfunction-based assays of activity that are generally known in the art.

Voltage-Gated Ion Channel Families

Voltage-Gated Sodium Channels (VGSCs) are expressed in membranes ofexcitable cells and function in the depolarization phase of actionpotentials. VGSCs expressed in mammalian brain tissues comprise poreforming and voltage sensing elements within a single polypeptide chain.There are nine known VGSC alpha subunits termed Na_(v)1.1 to Na_(v) 1.9which together make up a subfamily having about 70% amino acid sequenceidentity within their transmembrane segments (Goldin et al, 2000;Catterall et al. (2005) Pharmacol Rev 57:397-409).

Under resting conditions, the membrane potential of an excitable cell,as measured inside relative to outside, is polarized and can, in anon-limiting embodiment, be in the range of about −60 mV to about −80mV. Thus, at normal resting potentials, VGSCs will be in a closedconfirmation and be non-conducting. VGSCs generally adopt an openconformation upon membrane depolarization (about −40 mV) to allow a flowof sodium ions down a concentration gradient from the outside of thecell to the inside of the cell.

Voltage-Gated Calcium Channels (VGCCs) are similar to VGSCs and have analpha subunit analogous to the alpha subunit of sodium channels(Catterall et al. (2005) Pharmacol Rev 57:411-425). As with VGSCs, thealpha subunit of VGCCs is sufficient to form an ion selective pore byitself. There are been at least three VGCC alpha subunit subfamiliesdescribed in vertebrates. The Ca_(v)1 subfamily (Ca_(y) 1.1 to Ca_(v)1.4) conducts L-type (long-lasting) currents (Hoffmann at al, 1994;Streissnig, 1999). Agents which block calcium channels are known in theart. For example, phenylalkalamines, dihydropyridines, andbenzothiazepines bind to S5 and S6 segments in domain III and the S6segment in domain IV and block the function of Ca_(v) 1.2 channels. TheCa_(v)2 subfamily (Ca_(v) 2.1 to Ca_(v) 2.3) conduct N-, P/Q, and R-typecalcium currents. It is known that Ca_(v)2 subfamily members can beblocked with spider and cone snail venoms (Smith and Reiner, 1992,Dunlap et al, 1995; Catterall, 2000b; Olivera et al, 1994). The Ca_(v)3subfamily (Ca_(v) 3.1 to Ca_(v) 3.3) conduct T-type (transient) calciumcurrents (Perez-Reyes, 2003).

Voltage-Gated Potassium Channels (VGPCs) undergo activation in responseto membrane depolarization to promote outward movement of potassium ionsto allow the termination of action potentials through membranerepolarization. At least 40 different VGPC alpha subunits have beenidentified across 12 different VGPC subfamilies (Kv1 to Kv12) (Gutman etal, 2003; Gutman et al. (2005) Pharmacol Rev 57:473-508). The first VGPCcharacterized was cloned in Drosophila on the basis of a “Shaker”phenotype (Jan and Jan, 1997). Several types of potassium channels areknown in the art. These include potassium channels in cardiac muscle,including but not limited to inward rectifying K+ channels (IKr),delayed rectifying K+ channels (Kv or IKs) and transient outward K+channels (IKTo); many of these cardiac potassium channels belong to theVGPC family.

VGPCs comprise tetramers of alpha subunits wherein each alpha subunitcomprises six hydrophobic transmembrane segments (S1 to S6) and whereinthe alpha subunits are assemble around the central pore. Both VGSCs andVGCCs share sequence identity and structural similarity to Shakerpotassium channel members, however, as mentioned above, VGPCs are formedby homomeric or heteromeric alpha subunit tetramers, whereas in the caseof VGCCs and VGPCs, the channel pore is formed by a single alpha subunitcomprising four sequential pseudo-domains (each of which is homologousto a VGPC alpha subunit). The central ion conductive pore (the alphapore) of VGPCs is formed in alpha subunit tetramers by the regioncomprising the S5 and S6 segments of each alpha subunit within thechannel complex. Ion selectively of VGICs depends on a hydrophobic 20amino acid loop, the so-called “P-loop,” located between the S5 and S6segments. This ion selectivity loop is conserved between VGPCsubfamilies and is marked by a [T/S]-[M/L/Q]-T-T-[I/V]-G-Y-G signaturesequence (SEQ ID NO: 200). This S5-loop-S6 domain is termed the PoreDomain (PD).

Voltage-Gated Proton Channels (VGHCs) are expressed in the membranes ofmany cell types and appear to facilitate diverse physiologicalfunctions. For example, the human VGHC Hv1 is known to regulate reactiveoxygen species production by leukocytes, histamine secretion bybasophils, sperm capacitation, and airway pH. (Ramsey et al. (2006) Avoltage-gated proton-selective channel lacking the pore domain. Nature440:1213-1216).

Additional voltage-gated ion channels, all of which possess VSDsstructurally and functionally analogous to those discussed above,comprise the HCN and CatSper ion channel families, and certain membersof the KCa ion channel family.

The HCN channel family comprises HCN1, HCN2, HCN3, and HCN4 (Hofmann etal. (2005) “International Union of Pharmacology. LI. Nomenclature andStructure-Function Relationships of Cyclic Nucleotide-RegulatedChannels” Pharmacol Rev 57:455-462). HCN channels are found in neuronsand the heart; in the heart these channels control heart rate and rhythmby form the so-called “Ih” or “pacemaker current” in the sino-atrialnode; in neurons these channels help to determine resting potentials,the transduction of sour taste, and synaptic transmission andplasticity. Drugs that interact with HCN channels, for exampleivabradine and cilobradine, find use as heart rate-lowering agents inthe therapy of angina pectoris.

The CatSper channel family comprises CatSper1, CatSper2, CatSper3, andCatSper4 (Clapham & Garbers (2005) “International Union of Pharmacology.L. Nomenclature and Structure-Function Relationships of CatSper andTwo-Pore Channels” Pharmacol Rev 57:451-454). CatSper1 and CatSper2control intracellular Ca²⁺ levels in sperm cells; CatSper1- orCatSper2-null sperm cells cannot be hyperactivated, and these geneknockouts result in a male sterile phenotype. CatSper-interacting drugsmay find use as fertility modulating agents.

The BK (also known as Slo1 or MaxiK) channel, a member of the KCa ionchannel family, also contains VSDs which modulate the channel's ionconductance in response to membrane voltage (Wei et al. (2005)“International Union of Pharmacology. LII. Nomenclature and molecularrelationships of calcium-activated potassium channels” Pharmacol Rev57:463-472). The BK channel modulates diverse intracellular Ca²⁺signaling responses, and as such play diverse roles in normal physiologyand pathophysiology. For example, it has been found that alterations inBK channel function can cause hypertension (Holtzclaw et al. (2011)“Role of BK channels in hypertension and potassium secretion” Curr OpinNephrol Hypertens 20(5):512-517).

In VGSCs, VGCCs, and VGPCs, the ion permeability of the pore domain iscontrolled by the voltage sensor domains, which surround the centralpore domain. Opening of the pore domain occurs in a process whereintransmembrane voltage changes induce four conserved positively chargedresidues (e.g. arginine residues) in the S4 segment of each VSD in eachof the four alpha subunits (VGPCs), or the four pseudo-domains (VGSCsand VGCCs), to move outward across the membrane; complete outwardmovement all four S4 transmembrane segments in a channel subsequentlyinduces the pore domain to open, allowing it to conduct ions (see HilleB. (2001) Ion Channels of Excitable Membranes, 3rd Ed., pp. 131-143;Tombola et al., 2006) Annu. Rev. Cell Dev. Biol. 22, 23-52). Thisopening process is also known as “gating”. The ion current passingthrough the main pore is called the “alpha current”.

The pore-forming alpha subunits of channels in three of the four VGICfamilies, the VGSCs, the VGCCs, and the VGPCs, are comprised of foursubunits, or in some cases, four pseudo-domains that provide functionalsimilarity to subunits. VGPCs comprise four subunits, arranged either ashomotetramers or heterotetramers. VGSCs and VGCCs comprise a singlepolypeptide chain that contains within it four pseudo-domains; each ofthese pseudo-domains has high sequence homology in comparison to theother three pseudo-domains of that particular VGSC or VGCC, and incomparison to the pseudo-domains in other VGSCs or VGCCs, or the alphasubunits of VGPCs. The alpha subunits of VGPCs, or each pseudo-domain ofthe VGSCs and VGCCs alpha subunits, each comprise a conserved voltagesensing VSD (comprised of transmembrane S1, S2, S3, and S4 segments)followed in primary sequence by a conserved pore forming ion conductionpathway, the pore motif, wherein a narrow outer pore mouth comprisesso-called “P-loops” between the transmembrane S5 and S6 segments. VGSCs,VGCCs, and VGPCs are each distinguished from one another by certainconserved sequences in these P-loops, which appear to confer selectivityfor the transport of particular types of ions. VGICs can be classifiedby gating type and gating number.

In commonality with VGSC, VGCC, and VGPC alpha subunits, thevoltage-sensing elements of VGHCs comprises a VSD, again comprised ofconserved transmembrane 51, S2, S3, and S4 segments. In distinction tothe VGSC, VGCC, and VGPC alpha subunits, VGHCs lack a pore motif.Instead, the VSD itself appears to constitute the pore forming ionconduction pathway within VGHCs. Additionally, VGHCs appear to functionas dimers, not tetramers.

Gating in VGHCs occurs when transmembrane voltage changes induce fourconserved positively charged residues (e.g. arginine residues) in the S4segment of each VSD to outward across the membrane; it appears that bothS4 segments in VGHC dimers need to move (at least partly) outward toallow one or both VGHC VSDs to attain a conformation that allowsconduction of protons. Distinct from VGSCs, VGCCs, and VGPCs, however,VGHCs conduct protons directly through activated (or open) VSDs, ratherthan through a separate pore domain.

In the case of Shaker potassium channel members, the pore changesconformation to an inactivated state upon extended depolarization.N-type and C-type inactivation of Shaker potassium channel members isalso known.

Ion Channel Current Leak Amino Acid Substitutions

VGICs having defined mutations have revealed the existence of anotherion current, termed the omega leak current (i.e., omega pore dependention permeation). For example, substitution of arginine residues in theS4 helix of the Shaker channel with a smaller, typically polar,uncharged amino acid (e.g. serine or cysteine) results in an omega leakcurrent through the voltage sensing domains of the Shaker channel thatallows ion permeation independently of the normal alpha pore current(Tombola et al., Nature. 2007 Feb. 1; 445(7127):546-9). In the case ofrat or human Kv1.2 these arginine residues are located at positions 294,297, 300, and 303 of the protein. One of skill in the art will readilybe capable of identifying positively charged residues in the voltagesensing domain of other VGICs or other VGPs on the basis of sequenceidentity between VGICs. One of skill in the art will also understandthat certain classes of VGICs (e.g., VGCCs and VGSCs) can have fourdistinct VSDs, whereas other VGICs (e.g., VGPCs) have only one distinctVSD. A VGIC having an omega pore ion permeation can be a VGIC comprisingan omega pore ion permeation inducing amino acid substitution at one ormore positions in the VSD in a VGIC heterotetrameric complex (e.g. amixed tetrameric VGIC complex) or in a VGIC that is not aheterotetrameric complex (e.g. a homotetrameric VGIC complex).

The omega leak current passes through an omega pore located in thevoltage sensing domains (S1-S4) of the channel (Tombola et al., (2006)Annu. Rev. Cell Dev. Biol. 22, 23-52; Tombola et al., (2005) Neuron 45,379-388; Tombola et al., (2007) Nature 445, 546-549; Sokolov et al.,(2007) Nature 446, 76-78; Sokolov et al., (2005) Neuron 47, 183-189;Starace et al., (1997) Neuron. 19:1319-1327). Omega currents arenonselective and allow conduction of various cations. The omega leakcurrent can be observed by investigation under hyperpolarizingconditions when the S4 segment and the alpha pore are in the closedconfiguration. Omega leak currents can also be observed for VGICs havingcertain other defined VSD mutations; in this case, omega currents areobserved under depolarizing conditions when the S4 segment and the alphapore are in the open configuration (Sokolov et al., (2007) Nature 446,76-78; Sokolov et al., (2005) Neuron 47, 183-189). Observation of theselatter omega currents is facilitated by modulating experimentalconditions (e.g., through use of ion channel modulating agents) so as toblock the normal alpha current.

Another leak current, termed the “sigma current”, has also beenidentified in the Shaker-related human Kv1.3 channel. The sigma porepathway was identified by examining the conductance properties of a VGPChuman Kv1.3 mutant protein having a V to C mutation corresponding toDrosophila Shaker position 438 (Pruning and Grissmer, J Biol Chem. 2011Jun. 3; 286(22):20031-42). The sigma leak current appears to befunctionally similar to the omega leak currents described above. Themutation that induces sigma currents appears to be physically distinct,however, from those mutations that induce omega currents; the sigmacurrent mutation is located adjacent to the normal alpha pore, ratherthan being located within the VSD, as is the case for omegacurrent-inducing mutations. A VGIC having a sigma pore ion permeationcan be a VGIC comprising a sigma pore ion permeation inducing amino acidsubstitution at one or more positions adjacent to the normal alpha porein a VGIC heterotetrameric complex or in a VGIC that is not aheterotetrameric complex (e.g. a homotetrameric VGIC complex). Incertain embodiments, an alpha pore current or an omega pore current canbe more amenable to reliable measurement than a sigma pore current.

Other abnormal currents can be observed in VGHCs, for example the humanHv1 channel, in the presence of certain VSD mutations, in particularmutation of aspartate residue 112 (Berger and Isacoff, (2011) Neuron,27:991-1000; Musset et al., (2011), Nature, 480:273-277). Thesecurrents, which can be carried by various anions or cations depending onthe experimental conditions, also appear to flow through the mutatedVSD.

The methods described herein relate to the use of ion channelscomprising one or more mutations, wherein the mutations cause the ionchannel to exhibit ion permeability when the ion channel is in a closedconformation and/or when the ion channel is in an inactivatedconformation. In certain embodiments, the channels having closed and/orinactivated configuration permeability can be channels having omega poredependent ion permeation, or channels having sigma pore dependent ionpermeation.

Several mutations causing omega or sigma pore dependent ion permeationhave been identified in the art. For example, and without wishing to bebound by theory, in the case of the Kv2.1 ion channel, mutation ofconserved gating residues (e.g. arginine or lysine) residues in the S4helix of the protein can cause the multimeric forms of the protein toexhibit nonselective conductance of various cations in the form of anomega current, even when the Kv2.1 VGIC complex is in a closed orinactive conformation. Such mutations include, but are not limited to anR300Q, R303Q mutation, an R306Q mutation, or an R309Q mutation, or anycombination thereof in human Kv2.1.

Because VGIC proteins exhibit sequence identity and structuralsimilarity, one of skill in the will readily be capable of introducinganalogous mutations in other VGIC proteins so as to cause the otherVGICs to exhibit non-alpha pore dependent leaks. One of skill in the artwill readily be capable of identifying the S4 helix, and the arginineresidues therein, in other ion channels from different subfamilies,families and species using standard sequence comparison algorithms. Onealgorithm that can be used to identify gating residues in a ion channelsuitable for mutation to introduce a omega current in the ion channel inthe BLAST sequence alignment program, however, other programs suitablefor use to align two or more sequences according to primary sequenceand/or predicted or known secondary structural features of an ionchannel can also be used. For example, an alignment between the humanKv2.1 protein and the human Kv2.2 protein shows that the S4 helixresidues at positions R303, R306, and K309 of human Kv2.1 correspond topositions R307, R310 and K313 of the human Kv2.2 protein. Thus, one ofskill in the art will readily appreciate that introduction of an R307Qmutation, an R310Q mutation or a K313Q mutation will cause human Kv2.2to exhibit an omega leak current when it is in closed and/or inactivatedstate. Similar methods can be used to identify residues in other ionchannel proteins to generate ion channels exhibiting an omega leakcurrent in a closed and/or inactivated state. Additional positivelycharged S4 residues may need to be mutated, (e.g., R291 and R290 inK_(v)2.1) to enhance omega-pore dependent ion permeation.

Where the VGP is a VGIC, one or more mutations can be introduced intothe VGIC to cause the VGIC to exhibit a leak current when the ionchannel is in a closed conformation and/or when the ion channel is in aninactivated conformation. Alternatively, where the VGP is a VSP, one ormore mutations can be introduced into the VGIC to cause the VGIC toexhibit altered voltage sensitivity.

In certain embodiments, the mutation is an R to S or R to N mutation ata position within the S4 helix of the VGIC. Without wishing to be boundby theory, in certain embodiments, analogous mutations can be introducedto generate variants of VGICs described herein wherein one or morearginine residues in the S4 helix of the VGIC are substituted withanother amino acid having a non-polar side chain so as to mimic theeffect of the R to S or R to N mutation in the mutated VGIC. Amino acidresidues having similar side chain configurations have been defined inthe art within in accordance with the following categories: basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine), aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine), aliphatic sidechains (e.g., glycine, alanine, valine, leucine, isoleucine), andsulfur-containing side chains (methionine, cysteine). Substitutions canalso be made between acidic amino acids and their respective amides(e.g., asparagine and aspartic acid, or glutamine and glutamic acid).

Several mutations causing omega or sigma channel leaks have beenidentified in the art. For example, and without wishing to be bound bytheory, in the case of the Kv2.1 ion channel, mutation of conserved ornon-conserved gating residues (e.g. arginine or lysine residues) in theS4 helix of the protein can cause the multimeric forms of the protein toexhibit nonselective conductance of various cations in the form of anomega current, even when the Kv2.1 VGIC complex is in a closed orinactive conformation. Such mutations include, but are not limited to anR294N mutation or an R300S mutation, or any combination thereof in humanKv2.1. Thus, in one embodiment, an ion channel having an omega leaksuitable for use with the methods described herein will be a Kv2.1protein comprising an amino acid substitution at any of positions R300,R303, or R306, wherein the substitution replaces the positively chargedarginine residues with an amino acid substituent residue havinguncharged polar side chains wherein any of the arginine as positionsR300, R303 or R306 are replaced with any of glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine or glutamic acid. Oneof skill in the art will readily be capable of identifying othervariants of the mutated VGICs described herein. In one embodiment, themutations in Kv2.1 include, but are not limited to an R294N mutation, anR300S mutation, an R294C mutation, an R293S mutation, an R294S mutation,or any combination thereof. For example, human Kv2.1 can have mutationsR294C and R300S, mutations R293S and R294S, mutations R293S and R294C,mutations R293S, R294S, and R300S, or mutations R293S, R294C, and R300S.In one embodiment, a Kv2.1 protein can be mutated to removeextracellular cysteine residues. In some embodiments, the Kv2.1 proteinwith mutations R293S, R294S, and R300S can be used for drug screening.

Similarly, mutation of Drosophila Shaker R362 to histidine, forinstance, is associated with abnormal omega currents; these currents areH⁺ currents (Starace et al., Nature 427, 548-553 (2004)). Additionalmutations of Shaker R362 which give rise to omega currents, carried by,e.g., Na⁺ ions, have been characterized (Tombola et al., Neuron 45,379-388 (2005); Tombola et al., Nature 445, 546-549 (2007)). In oneembodiment, an ion channel having an omega leak suitable for use withthe methods described herein will be a Shaker protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R362, R365, R368, R371, K374, or R377,wherein the substitution replaces the positively charged arginineresidue with an amino acid substituent residue having an uncharged polarside chain or with an amino acid residue not having a positively chargedside chain. In one embodiment, the amino acid substitute at the gatingcharge position is a serine or an asparagine. In one embodiment, theamino acid substitute at the gating charge position is a serine or anasparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Nav1.1 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions K1313, R1316, R1319, R1322, or R1325,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Nav1.2 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions K1303, R1306, R1309, R1312 or R1315,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Nav1.3 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions K1301, R1304, R1307, R1310, or R1313,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Nav1.4 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions K1126, R1129, R1132, R1135 or R1137,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Nav1.5 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions K1300, R1303, R1306, R1309 and R1312,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Nav1.6 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions K1293, 1296, R1299, R1302 or R1305, whereinthe substitution replaces the positively charged arginine or lysineresidue with an amino acid substituent residue having an uncharged polarside chain or with an amino acid residue not having a positively chargedside chain. In one embodiment, the amino acid substitute at the gatingcharge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Nav1.7 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions K1276, R1279, R1282, R1285 or R1288,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Nav1.8 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions K1247, R1250, R1253, R1256, or R1259,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Nav1.9 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions K1144, R1147, R1150, R1153, or R1156,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Cav1.1 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions K912, or K915, wherein the substitutionreplaces the positively charged lysine residue with an amino acidsubstituent residue having an uncharged polar side chain or with anamino acid residue not having a positively charged side chain. In oneembodiment, the amino acid substitute at the gating charge position is aserine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Cav1.2 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions K984 or K987, wherein the substitutionreplaces the positively charged lysine residue with an amino acidsubstituent residue having an uncharged polar side chain or with anamino acid residue not having a positively charged side chain. In oneembodiment, the amino acid substitute at the gating charge position is aserine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Cav1.3 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions K999 or K1002 wherein the substitutionreplaces the positively charged lysine residue with an amino acidsubstituent residue having an uncharged polar side chain or with anamino acid residue not having a positively charged side chain. In oneembodiment, the amino acid substitute at the gating charge position is aserine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Cav1.4 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions K973 or K976, wherein the substitutionreplaces the positively charged lysine residue with an amino acidsubstituent residue having an uncharged polar side chain or with anamino acid residue not having a positively charged side chain. In oneembodiment, the amino acid substitute at the gating charge position is aserine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv1.1 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R292, R295, R298, R301, K304, or R307,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv1.2 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R294, R297, R300, R303, K306, or R309,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv1.3 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R364, R367, R370, R373, K376, or R379,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv1.4 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R444, R447, R450, R453, K456, or R459,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv1.5 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R400, R403, R406, R409, K412, or R415,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv1.6 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R342, R345, R348, R351, K354 or R357,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv1.7 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R278, R281, R284, R287, K290 or R293,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv1.8 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R341, R344, R347, R350, K353 or R356,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv2.1 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R300, R303, R306, K309 or R312, wherein thesubstitution replaces the positively charged arginine or lysine residuewith an amino acid substituent residue having an uncharged polar sidechain or with an amino acid residue not having a positively charged sidechain. In one embodiment, the amino acid substitute at the gating chargeposition is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv2.2 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R304, R307, R310, K313 or R316, wherein thesubstitution replaces the positively charged arginine or lysine residuewith an amino acid substituent residue having an uncharged polar sidechain or with an amino acid residue not having a positively charged sidechain. In one embodiment, the amino acid substitute at the gating chargeposition is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv3.1 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R311, R314, R317, R320, K323 or R326,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv3.2 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R348, R351, R354, R357, K360 or R363,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv3.3 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R414, R417, R420, R423, K426 or R429,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv3.4 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R347, R350, R353, R356, K359 or R362wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv4.1 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R295, R298, R301, K304, or R307, whereinthe substitution replaces the positively charged arginine or lysineresidue with an amino acid substituent residue having an uncharged polarside chain or with an amino acid residue not having a positively chargedside chain. In one embodiment, the amino acid substitute at the gatingcharge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv4.2 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R293, R296, R299, K302 or R305, wherein thesubstitution replaces the positively charged arginine or lysine residuewith an amino acid substituent residue having an uncharged polar sidechain or with an amino acid residue not having a positively charged sidechain. In one embodiment, the amino acid substitute at the gating chargeposition is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv4.3 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R292, R295, R298, K301 or R304, wherein thesubstitution replaces the positively charged arginine or lysine residuewith an amino acid substituent residue having an uncharged polar sidechain or with an amino acid residue not having a positively charged sidechain. In one embodiment, the amino acid substitute at the gating chargeposition is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv5.1 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R293, R296, K299, or R302, wherein thesubstitution replaces the positively charged arginine or lysine residuewith an amino acid substituent residue having an uncharged polar sidechain or with an amino acid residue not having a positively charged sidechain. In one embodiment, the amino acid substitute at the gating chargeposition is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv6.1 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R343, R346, R352 or R355, wherein thesubstitution replaces the positively charged arginine residue with anamino acid substituent residue having an uncharged polar side chain orwith an amino acid residue not having a positively charged side chain.In one embodiment, the amino acid substitute at the gating chargeposition is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv6.2 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R288, R291, R297 or R300, wherein thesubstitution replaces the positively charged arginine residue with anamino acid substituent residue having an uncharged polar side chain orwith an amino acid residue not having a positively charged side chain.In one embodiment, the amino acid substitute at the gating chargeposition is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv6.3 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R289, R292, R295, K301 or R304, wherein thesubstitution replaces the positively charged arginine or lysine residuewith an amino acid substituent residue having an uncharged polar sidechain or with an amino acid residue not having a positively charged sidechain. In one embodiment, the amino acid substitute at the gating chargeposition is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv6.4 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R337, R340, R343, R349 or R352, wherein thesubstitution replaces the positively charged arginine residue with anamino acid substituent residue having an uncharged polar side chain orwith an amino acid residue not having a positively charged side chain.In one embodiment, the amino acid substitute at the gating chargeposition is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv8.1 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R315, R318, R321, K324 or K327, wherein thesubstitution replaces the positively charged arginine or lysine residuewith an amino acid substituent residue having an uncharged polar sidechain or with an amino acid residue not having a positively charged sidechain. In one embodiment, the amino acid substitute at the gating chargeposition is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv8.2 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R377, R380, R383, R386, K389 or R392,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv9.1 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R345, R348, R351, K354 or R357, wherein thesubstitution replaces the positively charged arginine or lysine residuewith an amino acid substituent residue having an uncharged polar sidechain or with an amino acid residue not having a positively charged sidechain. In one embodiment, the amino acid substitute at the gating chargeposition is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv9.2 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R298, R301, R304, K307 or R310, wherein thesubstitution replaces the positively charged arginine or lysine residuewith an amino acid substituent residue having an uncharged polar sidechain or with an amino acid residue not having a positively charged sidechain. In one embodiment, the amino acid substitute at the gating chargeposition is a serine or an asparagine.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Kv9.3 protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R298, R301, R304, K307 or R310, wherein thesubstitution replaces the positively charged arginine or lysine residuewith an amino acid substituent residue having an uncharged polar sidechain or with an amino acid residue not having a positively charged sidechain. In one embodiment, the amino acid substitute at the gating chargeposition is a serine or an asparagine.

Other mutations outside of the S4 helix that cause an ion channel toexhibit an omega leak current in a closed and/or inactive configurationinclude those identified in Tombola et al, Nature 445, 546-549(2007).For example, the Drosophila shaker protein can be mutated to exhibit anomega leak current when in a closed and/or inactivated confirmation byintroducing one or more of the following mutations: V236C, E283D, C286S,F290C, A359G, Q354C, S357C, S352C, V453C, and W454C.

In one embodiment, an ion channel having an omega leak suitable for usewith the methods described herein will be a Shaker protein comprising anamino acid substitution of one or more S4 helix gating charge amino acidresidues at any of positions R362, R365, R368, R371, K374, or R377,wherein the substitution replaces the positively charged arginine orlysine residue with an amino acid residue having an uncharged polar sidechain or with an amino acid residue not having a positively charged sidechain. In one embodiment, the amino acid substitution at the gatingcharge position is a serine (e.g. R362S) or an asparagine (e.g. R362N).In one embodiment, the ion channel has an omega leak and an open alphapore. For example, the Drosophila Shaker protein can be mutated to havean omega leak and an open alpha pore by introducing the mutation R362S.Other mutations can cause an ion channel to have a closed alpha pore.For example, the Drosophila Shaker protein can be mutated to exhibit aclosed alpha pore by introduction of the mutation W434F. In oneembodiment, the ion channel has an omega leak and a closed alpha pore.For example, the Drosophila Shaker protein can be mutated to have anomega leak and a closed alpha pore by introducing the mutations R362Sand W434F. In some embodiments, the Shaker protein with mutations R362Scan be used for drug screening.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.1protein comprising at least one of the following amino acidsubstitutions: L911C, R1188D, C1191S, V1196C, A1302C, L1304C, S1307C,K1492C, or F1493C

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.2protein comprising at least one of the following amino acidsubstitutions: L902C, K1178D, C1188S, I1186C, N1192C, L1194C, S1197C,K1482C, or F1483C

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.3protein comprising at least one of the following amino acidsubstitutions: L903C, K1176D, F1179S, T1184C, N1190C, L1192C, S1195C,K1477C, or F1478C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.4protein comprising at least one of the following amino acidsubstitutions: L721C, R1001D, C1004S, I1009C, N1115C, L1117C, S1120C,K1304C, or L1305C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.5protein comprising at least one of the following amino acidsubstitutions: L860C, R1175D, C1178S, T1183C, N1298C, L1300C, A1303C,K1479C, or L1480C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.6protein comprising at least one of the following amino acidsubstitutions: L896C, R1168D, C1171S, I1176C, N1282C, L1284C, S1287C,K1473C, or F1474C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.7protein comprising at least one of the following amino acidsubstitutions: L876C, R1151D, C1154S, I1159C, N1265C, L1267C, S1270C,K1455C, or L1456C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.8protein comprising at least one of the following amino acidsubstitutions: L808C, H1123D, C1126S, T1130C, K1236C, L1238C, S1241C,A1243G, K1437C, or L1438C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.9protein comprising at least one of the following amino acidsubstitutions: L722C, G1022D, C1125S, D1033C, T1135C, L1137C, L1140C,M1141G, K1337C, or L1338C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav1.1protein comprising at least one of the following amino acidsubstitutions: N622C, F782D, T785S, K894C, L896C, L899C, N909G, I1114C,or V1115C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav1.2protein comprising at least one of the following amino acidsubstitutions: D685C, F854D, N857S, K966C, L968C, L971C, N981G, V1186C,or V1187C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav1.3protein comprising at least one of the following amino acidsubstitutions: D713C, L869D, T872S, K981C, L983C, L986C, N996G, V1201C,or V1202C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav1.4protein comprising at least one of the following amino acidsubstitutions: D708C, L843D, T846S, K955C, L953C, L956C, N966G, T1175C,or V1176C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav2.1protein comprising at least one of the following amino acidsubstitutions: D676C, L1230D, T1233S, K1346C, L1348C, L1351C, K1361G,F1565C, or V1566C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav2.2protein comprising at least one of the following amino acidsubstitutions: H671C, L1133D, T1136S, K1248C, L1250C, L1253C, K1263G,F1467C, or V1468C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav2.3protein comprising at least one of the following amino acidsubstitutions: N665C, F1117D, T1120S, K1236C, L1238C, L1241C, K1251G,F1455C, or V1456C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav3.1protein comprising at least one of the following amino acidsubstitutions: G827C, K974D, D977S, D990C, T1069C, S1071C, S1074C,M1083G, K1328C, or V1329C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav3.2protein comprising at least one of the following amino acidsubstitutions: D877C, G1022D, N1025S, T1028C, R1105C, S1107C, S1110C,Q1118G, K1346C, or V1347C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav3.3protein comprising at least one of the following amino acidsubstitutions: D689C, G834D, N837S, S840C, K916C, S918C, S921C, S926G,K1187C, or V1188C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.1protein comprising at least one of the following amino acidsubstitutions: V176C, E225D, C228S, F232C, Q284C, S287C, A289G, I383C,or G384C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.2protein comprising at least one of the following amino acidsubstitutions: V172C, E226D, C229S, F233C, Q286C, S289C, A291G, I385C,G386C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.3protein comprising at least one of the following amino acidsubstitutions: V243C, E299D, C302S, F306C, Q356C, S359C, A361G, I455C,or G456C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.4protein comprising at least one of the following amino acidsubstitutions: V316C, E375D, C378S, F382C, Q434C, Q436C, S439C, A441G,V555C, or G556C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.5protein comprising at least one of the following amino acidsubstitutions: V259C, E328D, C331S, F335C, G390C, Q392C, S395C, A397G,V491C, or G492C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.6protein comprising at least one of the following amino acidsubstitutions: V183C, E267D, C270S, F274C, G332C, Q334C, S337C, A339G,V433C, or G434C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.7protein comprising at least one of the following amino acidsubstitutions: V152C, E213D, C216S, F220C, G268C, Q270C, S237C, A275G,V369C, or G370C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.8protein comprising at least one of the following amino acidsubstitutions: V225C, E275D, C278S, F282C, A331C, Q333C, S336C, A338G,P432C, or G433C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv2.1protein comprising at least one of the following amino acidsubstitutions: D164C, C236S, F240C, L287C, F289C, V292C, R294G, L388C,or L389C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv2.2protein comprising at least one of the following amino acidsubstitutions: D168C, C240S, F244C, L291C, F293C, V296C, R298G, L392C,or L393C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv3.1protein comprising at least one of the following amino acidsubstitutions: F199C, C252S, F256C, K301C, A303C, V306C, W411C, orS412C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv3.2protein comprising at least one of the following amino acidsubstitutions: F238C, C289S, F293C, K338C, A340C, V343C, W448C, orS449C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv3.3protein comprising at least one of the following amino acidsubstitutions: F299C, C355S, F347C, K404C, A406C, V409C, W514C, orS515C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv3.4protein comprising at least one of the following amino acidsubstitutions: F235C, C285S, F292C, K337C, A339C, V347C, W447C, orS448C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv4.1protein comprising at least one of the following amino acidsubstitutions: H180C, V239S, F242C, K283C, D285C, S288C, A290G, I383C,or A384C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv4.2protein comprising at least one of the following amino acidsubstitutions: H179C, V237S, F240C, D281C, E283C, S286C, A288G, I381C,or A382C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv4.3protein comprising at least one of the following amino acidsubstitutions: H177C, V234S, F237C, N278C, E280C, S283C, A285G, I378C,or A379C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv5.1protein comprising at least one of the following amino acidsubstitutions: L191C, F233C, V285C, Q287C, Q290C, T381C, or L382C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv6.1protein comprising at least one of the following amino acidsubstitutions: G226C, C275S, F279C, Y334C, D336C, G339C, V341G, T435C,or P436C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv6.2protein comprising at least one of the following amino acidsubstitutions: G176C, C225S, F229C, L279C, E280C, G283C, V286G, L380C,or P381C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv6.3protein comprising at least one of the following amino acidsubstitutions: F180C, C228S, F232C, Q280C, Q282C, G285C, V286G, V384C,or P385C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv6.4protein comprising at least one of the following amino acidsubstitutions: G220C, C269S, F273C, Y328C, E330C, G333C, V335G, V429C,or P430C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv7.1protein comprising at least one of the following amino acidsubstitutions: V212C, E284C, K326C, I328C, C331C, F456C, or S457C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv7.2protein comprising at least one of the following amino acidsubstitutions: W270C, W344S, L393C, R395C, K398C, K537C, or V538C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv7.3protein comprising at least one of the following amino acidsubstitutions: W309C, W383S, A418C, S421C, Y541C, or D542C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv7.4protein comprising at least one of the following amino acidsubstitutions: W274C, W350S, V386C, R388C, N391C, T463C, or S464C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv7.5protein comprising at least one of the following amino acidsubstitutions: W304C, W378S, K428C, R430C, M433C, Y544C, or D545C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv8.1protein comprising at least one of the following amino acidsubstitutions: T191C, Y248S, F253C, G308C, I310C, V313C, T403C, orT404C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv8.2protein comprising at least one of the following amino acidsubstitutions: G240C, C309S, S368C, G370C, G373C, H468C, or L469C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv9.1protein comprising at least one of the following amino acidsubstitutions: S219C, C279S, F283C, H336C, G338C, V341C, V343G, V433C,or A434C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv9.2protein comprising at least one of the following amino acidsubstitutions: S186C, L240S, F246C, N289C, G291C, A294C, V296G, T386C,or A387C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv9.3protein comprising at least one of the following amino acidsubstitutions: S186C, L240S, F246C, N289C, G291C, A294C, V296G, T386C,or A387C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv10.1protein comprising at least one of the following amino acidsubstitutions: I281C, H391S, S435C, S437C, W440C, M563C, or R564C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv10.2protein comprising at least one of the following amino acidsubstitutions: I287C, H361S, T404C, A406C, W409C, M532C, or R533C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv11.1protein comprising at least one of the following amino acidsubstitutions: V483C, H562S, Y597C, S599C, L602C, L724C, or Q725C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv11.2protein comprising at least one of the following amino acidsubstitutions: V311C, H413S, Y448C, S450C, P453C, L576C, or Q577C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv11.3protein comprising at least one of the following amino acidsubstitutions: V482C, H564S, Y599C, D601C, S604C, L727C, or Q728C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv12.1protein comprising at least one of the following amino acidsubstitutions: 1288C, H368S, Y406C, G408C, T411C, L534C, or R535C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv12.2protein comprising at least one of the following amino acidsubstitutions: V292C, H372S, N428C, S430C, S433C, L443G, L565C, orR566C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv12.3protein comprising at least one of the following amino acidsubstitutions: 1294C, H374S, Y412C, N414C, V417C, L535C, or R536C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a HCN1protein comprising at least one of the following amino acidsubstitutions: V311C, T394S, Y434C, E436C, Y439C, T545C, or K546C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a HCN2protein comprising at least one of the following amino acidsubstitutions: V400C, T436S, Y503C, E505C, Y508C, T614C, or R615C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a HCN3protein comprising at least one of the following amino acidsubstitutions: V284C, T347S, Y387C, E389C, Y392C, T498C, or R499C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a HCN4protein comprising at least one of the following amino acidsubstitutions: V451C, T514S, Y554C, E556C, Y559C, T665C, or R666C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a CatSper1protein comprising at least one of the following amino acidsubstitutions: H214C, Y277S, Y313C, H315C, Y318C, P328G, W426C, orE427C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a CatSper2protein comprising at least one of the following amino acidsubstitutions: K239C, T289S, S334C, I336C, M339C, S433C, or K434C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a CatSper3protein comprising at least one of the following amino acidsubstitutions: L151C, F196C, K234C, L236C, R239C, T330C, or L331C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a CatSper4protein comprising at least one of the following amino acidsubstitutions: N146C, S229C, F231C, T234C, or E327C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Hv1 proteincomprising at least one of the following amino acid substitutions:V103C, K157C, F159C, R162C, S250C, or E251C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a KCa1.1protein comprising at least one of the following amino acidsubstitutions: G399C, D485S, P498C, Y593C, E595C, S598C, S610G, H787C,or V788C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a KCa4.1protein comprising at least one of the following amino acidsubstitutions: V500C, H586S, F635C, F637C, E640C, T809C, or A810C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a KCa4.2protein comprising at least one of the following amino acidsubstitutions: 1428C, H514S, F563C, N565C, Q568C, T727C, or A728C.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a TPC1protein comprising at least one of the following amino acidsubstitutions: S363C, N461S, L468C, Y520C, V522C, V525C, F355G, Y698C,or L699C.

VGIC function has been implicated in a variety of channelopathiesincluding, but not limited to, cardiac arrhythmias, familial periodicparalyses, cystic fibrosis, epilepsy, diabetes, asthma, angina pectoris,malignant hyperthermia, pain, hypertension, epilepsy, etc. Ion channelsrepresent key molecular targets for drug discovery. For example, Kv1.3channel function can play a role in T-cell immunosuppression, Kv2.1channel function can play a role in Type 2 diabetes, Kv1.5 channelfunction can play a role in Atrial fibrillation, Kv3.4 channel functioncan play a role in Alzheimer's disease, Kv7.1 channel function can playa role in atrial arrhythmia, Kv7.2 and Kv7.3 channel function can play arole in memory disorders, epilepsy, pain and migraine, Kv10.1 channelfunction can play a role in cancer, V-gated Na+/Ca2+ channels channelfunction can play a role in pain and hypertension, TRP channels channelfunction can play a role in pain and inflammation, CRAC channel functioncan play a role in T-cell immunosuppression. Thus, where mutationsassociated with VGIC function have been identified art, such mutants canbe used in connection with the methods described herein.

Several human channelopathies are typically associated with strictlyanalogous mutations in the gating charge arginine or lysine residueslocated in the S4-helix of the VSDs of various human VGSCs. Thesemutations allow omega dependent permeation when the channel is in theresting state, for certain mutations, or in the activated state, forcertain other mutations. Such gain-of-function mutants substantiallyincrease resting membrane conductance, leading to, for instance, cardiaclong QT syndromes, various paralyses, and migraine. One such example isthe R850Q/R853Q double mutant of the Nav1.2a channel; this doublemutation supports currents carried by K+, Cs+, Li+, Na+,tetramethylammonium, and tetraethylammonium, all in the presence of thealpha-pore blocker tetrodotoxin, thus demonstrating that these abnormalcurrents are omega currents (S. Sokolov, T. Scheuer, W. A. Catterall,Ion permeation through a voltage-sensitive gating pore in brain sodiumchannels having voltage sensor mutations. Neuron 47, 183-189 (2005)).Mutations in the Nav1.4 channel, including R663H, R666G, R666H, andR666S, are associated with hypokalaemic periodic paralysis; all thesemutations exhibit omega dependent permeation in the presence of thealpha-pore blocker tetrodotoxin (S. Sokolov, T. Scheuer, W. A.Catterall, Gating pore current in an inherited ion channelopathy. Nature446, 76-78 (2007)). Additional, similar mutations have been discussed(S. C. Cannon, Voltage-sensor mutations in channelopathies of skeletalmuscle. J. Physiol. 588, 1887-1895 (2010)), including those in Table 1.

TABLE 1 Gating charge mutations in ion channelopathies. Disease GeneChannel Mutation(s) Location HypoPP type I CACNA1S Cav1.1 R528H/G IIS4R1 R1239H/G IVS4 R2 HypoPP type II SCN4A Nav1.4 R669H IIS4 R1R672H/G/S/C IIS4 R2 K+-sensitive SCN4A Nav1.4 R675G/Q/W IIS4 R3Normokalemic PP HyperPP/Para- SCN4A Nav1.4 R1448C/H IVS4 R1 myotoniaCongenita Generalized SCN1A Nav1.l R859C IIS4 R1 Epilepsy with FebrileSeizures Plus R1648H IVS4 R5 Familial Hemi- CACNA1A Cav2.1 R192Q IS4 R1plegic Migraine/ Progressive Cerebellar Ataxia R583Q IIS4 R1 R1347QIIIS4 R1 Long QT 1 KCNQ1 KV7.1 R231C S4 R2 Long QT 2 KCNH2 KV11.1 K525NS4 R0 R528P S4 R1 Long QT 3 SCN5A Nav1.5 R225Q IS4 R3 R1623Q IVS4 R0The gating charge mutations in ion channelopathies are from thecollection of ion channelopathy mutations listed in the OMIM database athttp://www ncbi nlm nih.gov. Mutations that would alter an S4 gatingcharge in a voltage-gated Na+, Ca2+, or K+ channel were selected. 13 ofthe 16 disease mutations are in the R1 or R2 position in the S4 voltagesensor, and therefore would cause gating pore current in the restingstate of the ion channel like the HypoPP mutations studied here (S.Sokolov, T. Scheuer, W. A. Catterall, Gating pore current in aninherited ion channelopathy. Nature 446, 76-78 (2007)).

The channelopathy associated mutations shown in Table 1 are not intendedto be limiting and other channelopathy associated mutations capable ofinducing omega or sigma dependent permeation known in the art can beused in connection with the methods described herein. Further, becauseone of skill in the art will readily be capable of aligning the sequenceof any of the voltage-gated ion channel proteins shown in Table 1against other VGPs or VGICs, the methods described herein can also beperformed by generating a mutated VGIC protein comprising one or moresubstitutions analogous to the channelopathy associated mutations setforth in Table 1. Thus, in certain embodiments, the methods describedherein can be performed with a Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5,Nav1.6, Nav1.7, Nav1.8, Nav1.9, Cav1.1, Cav1.2, Cav1.3, Cav1.4, Cav2.1,Cav2.2, Cav2.3, Cav3.1, Cav3.2, Cav3.3, Kv1.1, Kv1.2, Kv1.3, Kv1.4,Kv1.5, Kv1.6, Kv1.7, Kv1.8, Kv2.1, Kv2.2, Kv3.1, Kv3.2, Kv3.3, Kv3.4,Kv4.1, Kv4.2, Kv4.3, Kv5.1, Kv6.1, Kv6.2, Kv6.3, Kv6.4, Kv7.1, Kv7.2,Kv7.3, Kv7.4, Kv7.5, Kv8.1, Kv8.2, Kv9.1, Kv9.2, Kv9.3, Kv10.1, Kv10.2,Kv11.1, Kv11.2, Kv11.3, Kv12.1, Kv12.2, Kv12.3, HCN1, HCN2, HCN3, HCN4,CatSper1, CatSper2, CatSper3, CatSper4, Hv1, KCa1.1, KCa4.1, KCa4.2,TPC1, Drosophila Shaker or any other VGIC or VGP comprising one or moreamino acid substitutions analogous to those set forth in Table 1 asdetermined by sequence alignment.

Other methods for generating ion channels having an omega leak currentinclude chemical modification of ion channels with thiol reactive agentssuch as ((2-trimethylammonium)ethyl)-methanethiosulphonate (MTSET) or(2-sulphonatoethyl)-methanethiosulphonate (MTSES). Without wishing to bebound by theory, MTSET and MTSES thiol reactive agents can modify ionchannels by adding bulk and either a positive or negative charge toresidues having a thiol group (e.g. cysteine). In the case of MTSET, theaddition will be bulk and a positive charge. In the case of MTSES, theaddition will be bulk and a negative charge. Mutations that can beintroduced into the Drosophila shaker ion channel that will render theion channel susceptible to MTSET treatment induced omega leak currentsare known in the art, and include, Q354C, S352C, V453C, M356C as well asother mutations described in Tombola et al, Nature 445, 546-549(2007).Mutations that can be introduced into the Drosophila shaker ion channelthat will render the ion channel susceptible to MTSES treatment inducedomega leak currents are known in the art, and include, Q354C, S352C,V453C, W454C as well as other mutations described in Tombola et al,Nature 445, 546-549(2007). Because one of skill in the art will readilybe capable aligning two or more amino acid sequences according toprimary sequence and/or predicted or known secondary structural featuresof an ion channel the residues corresponding to V236, E283, C286, F290,A359, 5352, Q354, M356, V453, W454 and other residues identified inTombola et al, Nature 445, 546-549(2007) can readily be mapped to otherion channel proteins to generate ion channels that exhibit omega leakcurrents either in the presence or absence of treatment with MTSET or inthe absence or presence of treatment with MTSES. For example, one ofskill in the art will appreciate that residues E283, C286, F290, A359,Q354 in the Drosophila shaker protein correspond to residues E229, C232.F236, Q284, respectively. Similar methods can be used to identifyresidues in other ion channel proteins to generate ion channelsexhibiting an omega leak current in a closed and/or inactivated stateeither in the presence or absence of treatment with MTSET or in theabsence or presence of treatment with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.1protein comprising any A1302C, L1304C, Y1306C, K1492C substitution orany combination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.2protein comprising any N1192C, L1194C, Y1196C, K1482C substitution orany combination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.3protein comprising any N1190C, L1192C, Y1194C, K1477C substitution orany combination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.4protein comprising any N1115C, L1117C, Y1119C, K1304C substitution orany combination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.5protein comprising any N1298C, L1300C, Y1302C, K1479C substitution orany combination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.6protein comprising any N1282C, L1284C, Y1286C, K1473C substitution orany combination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.7protein comprising any N1265C, L1267C, Y1269C, K1455C substitution orany combination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.8protein comprising any K1236C, L1238C, Y1240C, K1437C substitution orany combination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.9protein comprising any T1135C, L1137C, N1139C, K1337C substitution orany combination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav1.1protein comprising any K894C, L896C, V898C, I1114C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav1.2protein comprising any K966C, L968C, V970C, V1186C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav1.3protein comprising any K981C, L983C, V985C, V1201C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav1.4protein comprising any K955C, L957C, V959C, T1175C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav2.1protein comprising any K1346C, L1348C, V1350C, F1565C substitution orany combination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav2.2protein comprising any K1248C, L1250C, V1252C, F1467C substitution orany combination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav2.3protein comprising any K1236C, L1238C, V1240C, F1455C substitution orany combination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav3.1protein comprising any T1069C, S1071C, G1073C, K1328C substitution orany combination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav3.2protein comprising any R1105C, S1107C, S1109C, K1346C substitution orany combination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav3.3protein comprising any K916C, S918C, M920C, K1187C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.1protein comprising any Q284C, T286C, 1383C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.2protein comprising any Q286C, T288C, 1385C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.3protein comprising any Q356C, T358C, 1455C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.4protein comprising any Q434C, Q436C, M438C, V555C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.5protein comprising any G390C, Q392C, M394C, V491C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.6protein comprising any G332C, Q334C, M336C, V433C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.7protein comprising any G268C, Q270C, M272C, V369C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.8protein comprising any A331C, Q333C, M335C, P432C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv2.1protein comprising any L287C, F289C, N291C, L388C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv2.2protein comprising any L291C, F293C, N295C, L392C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv3.1protein comprising any K301C, A303C, D305C, W411C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv3.2protein comprising any K338C, A340C, D342C, W448C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv3.3protein comprising any K404C, A406C, D408C, W514C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv3.4protein comprising any K337C, A339C, D341C, W447C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv4.1protein comprising any K283C, D285C, V287C, I383C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv4.2protein comprising any D281C, E283C, V285C, I381C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv4.3protein comprising any N278C, E280C, V282C, I378C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv5.1protein comprising any V285C, Q287C, V289C, T381C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv6.1protein comprising any Y334C, D336C, V338C, T435C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv6.2protein comprising any L279C, E280C, A282C, L380C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv6.3protein comprising any Q280C, Q282C, A284C, V384C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv6.4protein comprising any Y328C, E330C, V332C, V429C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv7.1protein comprising any K326C, I328C, S330C, F456C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv7.2protein comprising any L393C, R395C, L397C, K537C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv7.3protein comprising any A418C, S420C, Y541C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv7.4protein comprising any V386C, R388C, R390C, T463C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv7.5protein comprising any K428C, R430C, R432C, Y544C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv8.1protein comprising any G308C, I310C, Q312C, T403C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv8.2protein comprising any S368C, G370C, V372C, H468C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv9.1protein comprising any H336C, G338C, V340C, V433C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv9.2protein comprising any N289C, G291C, V293C, T386C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv9.3protein comprising any N289C, G291C, V293C, T386C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv10.1protein comprising any S435C, S437C, K439C, M563C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv10.2protein comprising any T404C, A406C, 1408C, M532C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv11.1protein comprising any Y597C, S599C, G601C, L724C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv11.2protein comprising any Y448C, S450C, D452C, L576C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv11.3protein comprising any Y599C, D601C, D603C, L727C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv12.1protein comprising any Y406C, G408C, N410C, L534C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv12.2protein comprising any N428C, S430C, S432C, L565C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv12.3protein comprising any Y412C, N414C, S416C, L535C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a HCN1protein comprising any Y434C, E436C, R438C, T545C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a HCN2protein comprising any Y503C, E505C, R507C, T614C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a HCN3protein comprising any Y387C, E389C, R391C, T498C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a HCN4protein comprising any Y554C, E556C, R558C, T665C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a CatSper1protein comprising any Y313C, H315C, D317C, W426C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a CatSper2protein comprising any S334C, I336C, A338C, S433C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a CatSper3protein comprising any K234C, L236C, N238C, T330C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a CatSper4protein comprising any S229C, F231C, V233C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Hv1 proteincomprising any K157C, F159C, F161C, S250C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a KCa1.1protein comprising any Y593C, E595C, V597C, H787C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a KCa4.1protein comprising any F635C, F637C, Q639C, T809C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a KCa4.2protein comprising any F563C, N565C, D567C, T727C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a TPC1protein comprising any Y520C, V522C, A524C, Y698C substitution or anycombination thereof that has been treated with MTSET.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.1protein comprising any A1302C, L1304C, K1492C, F1493C substitution orany combination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.2protein comprising any N1192C, L1194C, K1482C, F1483C substitution orany combination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.3protein comprising any N1190C, L1192C, K1477C, F1478C substitution orany combination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.4protein comprising any N1115C, L1117C, K1304C, L1305C substitution orany combination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.5protein comprising any N1298C, L1300C, K1479C, L1480C substitution orany combination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.6protein comprising any N1282C, L1284C, K1473C, F1474C substitution orany combination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.7protein comprising any N1265C, L1267C, K1455C, L1456C substitution orany combination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.8protein comprising any K1236C, L1238C, K1437C, L1438C substitution orany combination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Nav1.9protein comprising any T1135C, L1137C, K1337C, L1338C substitution orany combination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav1.1protein comprising any K894C, L896C, I1114C, V1115C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav1.2protein comprising any K966C, L968C, V1186C, V1187C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav1.3protein comprising any K981C, L983C, V1201C, V1202C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav1.4protein comprising any K955C, L953C, T1175C, V1176C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav2.1protein comprising any K1346C, L1348C, F1565C, V1566C substitution orany combination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav2.2protein comprising any K1248C, L1250C, F1467C, V1468C substitution orany combination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav2.3protein comprising any K1236C, L1238C, F1455C, V1456C substitution orany combination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav3.1protein comprising any T1069C, S1071C, K1328C, V1329C substitution orany combination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav3.2protein comprising any R1105C, S1107C, K1346C, V1347C substitution orany combination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Cav3.3protein comprising any K916C, S918C, K1187C, V1188C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.1protein comprising any Q284C, I383C, G384C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.2protein comprising any Q286C, 1385C, G386C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.3protein comprising any Q356C, 1455C, G456C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.4protein comprising any Q434C, Q436C, V555C, G556C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.5protein comprising any G390C, Q392C, V491C, G492C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.6protein comprising any G332C, Q334C, V433C, G434C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.7protein comprising any G268C, Q270C, V369C, G370C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv1.8protein comprising any A331C, Q333C, P432C, G433C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv2.1protein comprising any L287C, F289C, L388C, L389C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv2.2protein comprising any L291C, F293C, L392C, L393C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv3.1protein comprising any K301C, A303C, W411C, S412C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv3.2protein comprising any K338C, A340C, W448C, S449C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv3.3protein comprising any K404C, A406C, W514C, S515C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv3.4protein comprising any K337C, A339C, W447C, S448C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv4.1protein comprising any K283C, D285C, I383C, A384C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv4.2protein comprising any D281C, E283C, I381C, A382C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv4.3protein comprising any N278C, E280C, I378C, A379C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv5.1protein comprising any V285C, Q287C, T381C, L382C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv6.1protein comprising any Y334C, D336C, T435C, P436C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv6.2protein comprising any L279C, E280C, L380C, P381C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv6.3protein comprising any Q280C, Q282C, V384C, P385C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv6.4protein comprising any Y328C, E330C, V429C, P430C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv7.1protein comprising any K326C, I328C, F456C, S457C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv7.2protein comprising any L393C, R395C, K537C, V538C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv7.3protein comprising any A418C, Y541C, D542C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv7.4protein comprising any V386C, R388C, T463C, S464C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv7.5protein comprising any K428C, R430C, Y544C, D545C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv8.1protein comprising any G308C, I310C, T403C, T404C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv8.2protein comprising any S368C, G370C, H468C, L469C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv9.1protein comprising any H336C, G338C, V433C, A434C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv9.2protein comprising any N289C, G291C, T386C, A387C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv9.3protein comprising any N289C, G291C, T386C, A387C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv10.1protein comprising any S435C, S437C, M563C, R564C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv10.2protein comprising any T404C, A406C, M532C, R533C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv11.1protein comprising any Y597C, S599C, L724C, Q725C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv11.2protein comprising any Y448C, S450C, L576C, Q577C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv11.3protein comprising any Y599C, D601C, L727C, Q728C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv12.1protein comprising any Y406C, G408C, L534C, R535C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv12.2protein comprising any N428C, S430C, L565C, R566C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Kv12.3protein comprising any Y412C, N414C, L535C, R536C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a HCN1protein comprising any Y434C, E436C, T545C, K546C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a HCN2protein comprising any Y503C, E505C, T614C, R615C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a HCN3protein comprising any Y387C, E389C, T498C, R499C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a HCN4protein comprising any Y554C, E556C, T665C, R666C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a CatSper1protein comprising any Y313C, H315C, W426C, E427C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a CatSper2protein comprising any S334C, I336C, S433C, K434C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a CatSper3protein comprising any K234C, L236C, T330C, L331C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a CatSper4protein comprising any S229C, F231C, E327C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a Hv1 proteincomprising any K157C, F159C, S250C, E251C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a KCa1.1protein comprising any Y593C, E595C, H787C, V788C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a KCa4.1protein comprising any F635C, F637C, T809C, A810C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a KCa4.2protein comprising any F563C, N565C, T727C, A728C substitution or anycombination thereof that has been treated with MTSES.

In one embodiment, an ion channel having omega pore dependent permeationsuitable for use with the methods described herein will be a TPC1protein comprising any Y520C, V522C, Y698C, L699C substitution or anycombination thereof that has been treated with MTSES.

Mutation of the valine corresponding to position 438 in the DrosophilaShaker protein is known to induce a sigma current in human Kv1.3channels 438 (Pruning and Grissmer, J Biol Chem. 2011 Jun. 3;286(22):20031-42). Sigma pore dependent ion permeation is similar toomega pore dependent ion permeation, however, unlike omega leak inducingmutations, mutations inducing sigma pore leaks are located adjacent tothe alpha pore, rather that being located within the VSD.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Shakerprotein comprising a V438C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Nav1.1protein comprising a V1442C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Nav1.2protein comprising a V1432C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Nav1.3protein comprising a V1427C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Nav1.4protein comprising a V1254C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Nav1.5protein comprising a V1429C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Nav1.6protein comprising a V1423C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Nav1.7protein comprising a V1405C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Nav1.8protein comprising a V1377C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Nav1.9protein comprising a V1267C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Cav1.1protein comprising a N1037C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Cav1.2protein comprising a Y1109C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Cav1.3protein comprising a H1124C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Cav1.4protein comprising a Y1098C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Cav2.1protein comprising a Y1486C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Cav2.2protein comprising a Y1388C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Cav2.3protein comprising a N1376C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Cav3.1protein comprising a G1221C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Cav3.2protein comprising a D1244C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Cav3.3protein comprising a K1068C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv1.1protein comprising a V368C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv1.2protein comprising a V370C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv1.3protein comprising a V440C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv1.4protein comprising a V520C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv1.5protein comprising a V476C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv1.6protein comprising a V418C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv1.7protein comprising a V354C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv1.8protein comprising a V417C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv2.1protein comprising a I373C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv2.2protein comprising a I377C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv3.1protein comprising a V396C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv3.2protein comprising a V433C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv3.3protein comprising a V499C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv3.4protein comprising a V432C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv4.1protein comprising a V368C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv4.2protein comprising a V366C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv4.3protein comprising a V363C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv6.1protein comprising a 1420C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv6.2protein comprising a I365C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv6.3protein comprising a W365C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv6.4protein comprising a I414C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv7.1protein comprising a K427C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv7.2protein comprising a F496C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv7.3protein comprising a R500C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv7.4protein comprising a S444C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv7.5protein comprising a Y500C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv8.1protein comprising a T388C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv8.2protein comprising a V453C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv9.1protein comprising a V417C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv9.2protein comprising a V370C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv9.3protein comprising a V370C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv10.1protein comprising a V519C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv10.2protein comprising a V488C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv12.1protein comprising a L490C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv12.2protein comprising a L521C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Kv12.3protein comprising a L495C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a HCN1protein comprising a V519C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a HCN2protein comprising a V588C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a HCN3protein comprising a L472C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a HCN4protein comprising a V639C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a CatSper1protein comprising a T406C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a CatSper2protein comprising a Y414C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a CatSper3protein comprising a A315C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a CatSper4protein comprising a M314C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a Hv1 proteincomprising a L231C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a KCa1.1protein comprising a M748C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a KCa4.1protein comprising a K751C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a KCa4.2protein comprising a K669C substitution.

In one embodiment, an ion channel having sigma pore dependent permeationsuitable for use with the methods described herein will be a TPC1protein comprising a R682C substitution.

The S2 segment of the Shaker channel comprises a well-conserved F290aromatic residue. This residues faces the intracellular side of thehydrophobic plug and forms part of a hydrogen bonding network withconserved basic residues in S2 and S3 in certain VGICs (Lacroix andBenzanilla, Proc Natl Acad Sci USA. 2011 Apr. 19; 108(16):6444-9). TheF290 residue in the Shaker protein controls the gating charge transitionenergy barrier. Substitution of the phenylalanine at position 290 of theShaker protein with a less hydrophobic residue produces fasterdeactivation kinetics relative to activation, wherein substitution witha more hydrophobic residue results slower deactivation kinetics relativeto activation (Tao et al., Science. 2010 Apr. 2; 328(5974):67-73). Asused herein, the hydrophobicity of an amino acid in a VGIC is measuredaccording to the hydrophobicity scale set forth in Wimley and White NatStruct Biol 1996, 3:842-848, wherein amino acid hydrophobicity, fromleast hydrophobic to most hydrophobic isE→D→K→R→H→Q→P→N→H→A→T→S→V→G→M→C→I→L→Y→F→W.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aShaker protein comprising a substitution of the phenylalanine atposition 290 with an amino acid that is less hydrophobic thanphenylalanine wherein the substituted protein has faster deactivationkinetics relative to activation or a substitution of the phenylalanineat position 290 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aCav 3.3 protein comprising a substitution of the tyrosine at position840 with an amino acid that is less hydrophobic than tyrosine whereinthe substituted protein has faster deactivation kinetics relative toactivation or a substitution of the tyrosine at position 840 with anamino acid that is more hydrophobic than tyrosine wherein thesubstituted protein has slower deactivation kinetics relative toactivation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv1.1. protein comprising a substitution of the phenylalanine atposition 232 with an amino acid that is less hydrophobic thanphenylalanine wherein the substituted protein has faster deactivationkinetics relative to activation or a substitution of the phenylalanineat position 232 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv1.2 protein comprising a substitution of the phenylalanine at position233 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 233 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv1.3 protein comprising a substitution of the phenylalanine at position308 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 308 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv1.4 protein comprising a substitution of the phenylalanine at position382 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 382 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv1.5 protein comprising a substitution of the phenylalanine at position335 with an amino acid that is less hydrophobic than tyrosine whereinthe substituted protein has faster deactivation kinetics relative toactivation or a substitution of the phenylalanine at position 335 withan amino acid that is more hydrophobic than phenylalanine wherein thesubstituted protein has slower deactivation kinetics relative toactivation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv1.6 protein comprising a substitution of the phenylalanine at position274 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 274 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv1.7 protein comprising a substitution of the phenylalanine at position220 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 220 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv1.8 protein comprising a substitution of the phenylalanine at position282 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 282 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv2.1 protein comprising a substitution of the phenylalanine at position240 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 240 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv2.2 protein comprising a substitution of the phenylalanine at position244 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 244 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv3.1 protein comprising a substitution of the phenylalanine at position256 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 256 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv3.2 protein comprising a substitution of the phenylalanine at position293 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 293 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv3.3 protein comprising a substitution of the tyrosine at position 359with an amino acid that is less hydrophobic than tyrosine wherein thesubstituted protein has faster deactivation kinetics relative toactivation or a substitution of the tyrosine at position 359 with anamino acid that is more hydrophobic than tyrosine wherein thesubstituted protein has slower deactivation kinetics relative toactivation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv3.4 protein comprising a substitution of the phenylalanine at position292 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 292 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv4.1 protein comprising a substitution of the phenylalanine at position242 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 242 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv4.2 protein comprising a substitution of the phenylalanine at position240 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 240 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv4.3 protein comprising a substitution of the phenylalanine at position237 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 237 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv5.1 protein comprising a substitution of the phenylalanine at position233 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 233 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv6.1 protein comprising a substitution of the phenylalanine at position279 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 279 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv6.2 protein comprising a substitution of the phenylalanine at position229 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 229 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv6.3 protein comprising a substitution of the phenylalanine at position232 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 232 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv6.4 protein comprising a substitution of the phenylalanine at position273 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 273 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv8.1 protein comprising a substitution of the phenylalanine at position253 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 253 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv9.1 protein comprising a substitution of the phenylalanine at position283 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 283 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv9.2 protein comprising a substitution of the phenylalanine at position246 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 246 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv9.3 protein comprising a substitution of the phenylalanine at position246 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 246 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv10.1 protein comprising a substitution of the tyrosine at position 398with an amino acid that is less hydrophobic than tyrosine wherein thesubstituted protein has faster deactivation kinetics relative toactivation or a substitution of the tyrosine at position 398 with anamino acid that is more hydrophobic than tyrosine wherein thesubstituted protein has slower deactivation kinetics relative toactivation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv10.2 protein comprising a substitution of the tyrosine at position 268with an amino acid that is less hydrophobic than tyrosine wherein thesubstituted protein has faster deactivation kinetics relative toactivation or a substitution of the tyrosine at position 268 with anamino acid that is more hydrophobic than tyrosine wherein thesubstituted protein has slower deactivation kinetics relative toactivation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv11.1 protein comprising a substitution of the tyrosine at position 569with an amino acid that is less hydrophobic than tyrosine wherein thesubstituted protein has faster deactivation kinetics relative toactivation or a substitution of the tyrosine at position 569 with anamino acid that is more hydrophobic than tyrosine wherein thesubstituted protein has slower deactivation kinetics relative toactivation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv11.2 protein comprising a substitution of the tyrosine at position 420with an amino acid that is less hydrophobic than tyrosine wherein thesubstituted protein has faster deactivation kinetics relative toactivation or a substitution of the tyrosine at position 420 with anamino acid that is more hydrophobic than tyrosine wherein thesubstituted protein has slower deactivation kinetics relative toactivation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv11.3 protein comprising a substitution of the tyrosine at position 571with an amino acid that is less hydrophobic than tyrosine wherein thesubstituted protein has faster deactivation kinetics relative toactivation or a substitution of the tyrosine at position 571 with anamino acid that is more hydrophobic than tyrosine wherein thesubstituted protein has slower deactivation kinetics relative toactivation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv12.1 protein comprising a substitution of the tyrosine at position 375with an amino acid that is less hydrophobic than tyrosine wherein thesubstituted protein has faster deactivation kinetics relative toactivation or a substitution of the tyrosine at position 375 with anamino acid that is more hydrophobic than tyrosine wherein thesubstituted protein has slower deactivation kinetics relative toactivation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv12.2 protein comprising a substitution of the phenylalanine atposition 379 with an amino acid that is less hydrophobic thanphenylalanine wherein the substituted protein has faster deactivationkinetics relative to activation or a substitution of the phenylalanineat position 379 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aKv12.3 protein comprising a substitution of the tyrosine at position 381with an amino acid that is less hydrophobic than tyrosine wherein thesubstituted protein has faster deactivation kinetics relative toactivation or a substitution of the tyrosine at position 381 with anamino acid that is more hydrophobic than tyrosine wherein thesubstituted protein has slower deactivation kinetics relative toactivation.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aCatsper3 protein comprising a substitution of the phenylalanine atposition 196 with an amino acid that is less hydrophobic thanphenylalanine wherein the substituted protein has faster deactivationkinetics relative to activation or a substitution of the phenylalanineat position 196 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

Ion Channel Modulating Agents

The methods described herein can also be performed with the use of smallmolecule agents that block or modulate the function of ion channels. Incertain embodiments, the methods described herein can be performed bycontacting an ion channel with a small molecule compound in the presenceor absence of a ion channel modulating agent.

Several such ion channel modulating agents are known in the art andinclude, but are not limited to turret blocking agents, main-poreblocking agents, gating-modifying agents, cysteine-tethered reagentagents, or voltage sensing domain toxins, including, but not limited tothose described in Triggle et al., Voltage-Gated Ion Channels as DrugTargets. Feb. 28, 2006 20:55, and Dilly et al., Chembiochem. 2011 Aug.16; 12(12):1808-12. Exemplary ion channel modulating agents suitable foruse with methods described herein include, but are not limited toclassical Kv channel inhibitors (e.g. 4-aminopyridine andtetramethylammonium), non specific compounds (e.g. calcium activatedpotassium channel blockers quinine and ceteidil), phenothiazineantipsychotics (e.g. chloropromazine and trifluoroperazine), classicalcalcium channel inhibitors (e.g. verapamil, diltiazem, nifedipine andnitrendipine) and beta blockers (e.g. propranolol). Other ion channelmodulating agents that can be used in connection with the methodsdescribed herein include, but are not limited to, iminodihydroquinolinesWIN173173 and CP339818 (Nguyen et al., 1996), the benzhydryl piperidineUK-78,282 (Hanson et al. 1999), correolide (Felix et al., 1999),cyclohexyl-substituted benzamide PAC (U.S. Pat. No. 6,194,458,WO0025774), sulfamidebenzamidoindane (U.S. Pat. No. 6,083,986),Khellinone (Baell et al., 2004), dichloropenylpyrazolopyrimidine(WO-00140231) psoralens (Wulff et al., 1998, Vennekamp et al., 2004,Schmitz et al., 2005) and isoindolines (WO 2008/038051). Table 2provides an exemplary list of ion channel modulating agents capable ofmodulating Kv1.3 ion conductance as well as the IC50 for each moleculeor metal.

TABLE 2 Exemplary ion channel modulating agents capable of modulatingKv1.3 ion conductance Ion Channel Ion Channel Modulating ModulatingAgents IC50 Agents IC50 PAP-1 2 nM H37 10 μM Psora-4 3 nM Hg2+ 10 μMTetraphenyl- 20 nM Kokusagenine 10 μM porphyrin 3 Correolide 30 nMQunine 14 μM C18-analog 43 trans-N-Propyl- 50 nM Cicutotoxin 18 μMcarbamoyloxy-PAC Correolide 90 nM Trifluoperazine 20 μMSulfamibenzamido- 100 nM Forskolin 20 μM indane CP-339818 150 nMCapsaisin 26 μM WIN-17317-3 200 nM Diltiazem 27 μM UK-78282 200 nMProgesterone 30 μM PAC 270 nM Luteolin 30 μM Khellinone dimer 2 280 nMLa3+ 50 μM Khellinone chalcone 16 400 nM Flecainide 60 μM6-(2-5-Dimethyl- 700 nM K22-Y23-R11 95 μM phenyl)psoralem ShK mimeticH-98 1.7 μM S-MOP 101 μM Reviniferatoxin 3 μM H2O2 100 μMPhenyl-stillbene A 2.9 μM 4-AP 195 μM Nifedipene 5 μM Zn2+, Co2+ 200 μMNitrendipene 5 μM Melatonin 1.5 mM Ibu-8 5 μM Ba2+, Cd2+ 2 mMPhenycylidine 5 μM TEA 10 mM Fluoxetine 6 μM Mn2+ 20 mM Varapamil 6 μM

Other ion channel modulating agents are known in the art and include,but are not limited to, lanthanum (III) chloride heptahydrate, which canbe dissolved to generate La³⁺ ions. In one embodiment, La³⁺ ions can beapplied by a fast perfusion technique. In one embodiment, La³⁺ ions canblock omega currents of an ion channel or ion channel mutant (e.g.Shaker channel, Kv2.1 channel). In one embodiment, La³⁺ ions do notblock omega currents of an ion channel or ion channel mutant (e.g.Shaker channel, Kv2.1 channel).

Other ion channel modulating agents are known in the art and alsoinclude, but are not limited to, 2-guanidinium-benzimidazole (2GBI),5-[(cyclopentylcarbonyl)amino]-2-(dimethylamino)-N-[(1R)-1-phenylethyl]-benzamide(B1), and3-methoxy-β-methyl-N-[2-(4-thiazolyl)-1H-benzimidazol-6-yl]-benzenepropanamide(RY785). In one embodiment, 2GBI, B1, or RY785 is used at a concentratonabove the IC50 for inhibition of the outward current through the alphapore. In one embodiment, 2GBI is used at a concentraton of 10 mM. In oneembodiment, B1 is used at a concentraton of 10 μM. In one embodiment,RY785 is used at a concentraton of 10 μM. In one embodiment, 2GBI, B1,or RY785 ions block omega currents of an ion channel or ion channelmutant (e.g. Shaker channel, Kv2.1 channel). In one embodiment, 2GBI,B1, or RY785 ions do not block omega currents of an ion channel or ionchannel mutant (e.g. Shaker channel, Kv2.1 channel).

Lanthanum (III) chloride heptahydrate, which can be dissolved togenerate La³⁺ ions. In one embodiment, La³⁺ ions can be applied by afast perfusion technique. In one embodiment, La³⁺ ions can block omegacurrents of an ion channel or ion channel mutant (e.g. Shaker channel,Kv2.1 channel). In one embodiment, La³⁺ ions do not block omega currentsof an ion channel or ion channel mutant (e.g. Shaker channel, Kv2.1channel).

Further ion channel modulating agents are known in the art and include,but are not limited to, cysteine-reactive reagents, including, but notlimited to MTSEA, MTSES, and MTSET. In one embodiment, acysteine-reactive reagent (e.g. MTSEA, MTSES, MTSET) can be applied by afast perfusion technique. In one embodiment, a cysteine-reactive reagent(e.g. MTSEA, MTSES, MTSET) can block inward gating pore leak currents ofan ion channel or ion channel mutant (e.g. Shaker channel, Kv2.1channel). In one embodiment, a cysteine-reactive reagent (e.g. MTSEA,MTSES, MTSET) do not block inward gating pore leak currents of an ionchannel or ion channel mutant (e.g. Shaker channel, Kv2.1 channel).

In certain embodiments, the methods described herein relate to themonitoring the activity of a mutated ion channel protein, wherein themutation causes the ion channel to exhibit an ion leak through eitherone or more omega or sigma pores when the channel is in a closed orinactivated state. In certain embodiments, the ion channel describedherein can comprise a mutation that renders the ion channel incapable,or partially incapable of adopting a closed or inactivated configurationsuch that ions remain capable of passing through an alpha pore of theion channel when the ion channel is subjected to a condition that willcause the channel to adopt either a closed or inactivated state. Thus,in embodiments, where an ion channel comprising a mutation that rendersthe channel incapable of blocking ion transit through the alpha pore ofthe on channel under closing or inactivating conditions, the methodsdescribed herein can further comprise a step of contacting the ionchannel in a membrane with one or more channel blocking toxins capableof blocking the alpha pore of the ion channel. Accordingly, channelblocking toxins can be used in connection with the methods describedherein to eliminate a contaminating current in an ion channel ofinterest. In certain embodiments, the contaminating current will be dueto the activity of a mutant channel under study. In certain embodiments,the contaminating current will be from one or more ion channelsendogenously expressed in a membrane of interest.

Many channel blocking toxins are known to exhibit specificity to aparticular type or class of ion channels. Further, the specificity ofthe particular channel blocking toxin can be concentration dependent.For example, it is known that tetrodotoxin (TTX) at a concentration of100 nM can be used to block the activity TTX sensitive VGSCs, but notaffect the activity of TTX insensitive VGSCs.

A number of channel blocking toxins are known in the art. For example,many peptides that are naturally occurring in scorpions, snakes andother marine organisms are known to be potent blockers of ion channels.

Charybdotoxin, originally isolated in the venom of the Leiurusquinquestriatus hebraeus scorpion, is on example of a channel blockingtoxin suitable for use with the methods described herein.

Other channel blocking toxins suitable for use for blocking the activityof an ion channel include, but are not limited to, simple pore blockersas well as gating modifiers. Without wishing to be bound by theory, poreblockers exhibit their effects on ion channel function by physicallyoccluding the entry of ions into the transmembrane pore, whereas gatingmodifiers allosterically inhibit structural rearrangements that areinvolved in channel activation. Pore blockers generally function bybinding to amino acid residues in the pore turret (as well as residueslocated close the pore turret) of voltage-gated ion channels (MacKinnonand Miller, 1989; MacKinnon et al., 1990; Gross et al., 1994; Gross andMacKinnon, 1996). Conversely, gating modifiers generally bind toconserved residues and structures on the extracellular face of thevoltage sensor. In the case of VGPCs and VGCCs, gating modifiersgenerally bind to the extracellular end of the S3 helix (Swartz andMacKinnon, 1997; Bourinet et al., 1999; Winterfield and Swartz, 2000).Without wishing to be bound by theory, when bound by a gating modifier,the ion channel will have a compromised ability to undergo structuralrearrangements required for activation of the channel and willaccordingly remain stability in an inactivated or a closed state.

Examples of pore blockers suitable for use with the methods describedherein include, but are not limited to, agitoxin, charybdotoxin, andω-conotoxin-GVI MacKinnon et al., 1990; Ellinor et al., 1994; Garcia etal., 1994; Feng et al., 2001). Examples of gating modifiers suitable foruse with the methods described herein include, but are not limited tohanatoxin, grammotoxin, and ω-agatoxin-IVA (Mintz et al., 1992; Lampe etal., 1993; Swartz and MacKinnon, 1995).

Other toxins include Guangxitoxin (GxTx-1E), a neurotoxin isolated fromPlesiophrictus guangxiensis venom. In one embodiment, GxTx-1E is used ata concentraton above the IC50 for inhibition of the outward currentthrough the alpha pore. In one embodiment, GxTx-1E is used at aconcentraton of 100 nM. In one embodiment, GxTx-1E can open a channelomega pore of an ion channel or ion channel mutant (e.g. Shaker channel,Kv2.1 channel).

In certain embodiments, the methods described herein can comprise a stepof contacting an ion channel to be monitored with the sea anemonestichodactyla helianthus toxin (Shk). Shk, and Shk derivatives (e.g.Shk-Dap²²) are potent Kv1.3 blockers that exhibit picomolar activity(Pennington et al, 1996; U.S. Pat. No. 6,077,680). Table 3 provides alist of channel blockers along with known IC50 values for Kv1.3 for anumber of ion channel modulating agents.

TABLE 3 List of ion channel modulating agents along with known IC50values IC50 Values of Kv1.3 ion Ion channel modulating agents channelmodulating agent OSK1-Lys16Asp20 3 pM Stichodactyla helianthus toxin(ShK) 0.9 pM to 110 pm Heterometrus spinnifer toxin 1 (HsTX1) 12 pMOrthochirus scrobiculosus toxin (OSK1) 14 pM Shk-F6CA 48 pM Pandinusimperator toxin 2 (Pi2) 50 pM Shk-Dap22 52 pM ShK(L5) 69 pM Hongotoxin(HgTX1) 86 pM Margatoxin (MgTX) 11 pM Agitotoxin-2 (AgTX2) 4 pM Pandinusimperator toxin 3 (Pi3) 500 pM Kaliolotoxin (KTX) 650 pM Anuroctoxin 730pM Noxiustoxin (NTX) 1 nM Charybdotoxin (ChTX) 0.19 nMTityustoxin-Kalpha (TsTX-Kalpha) 4 nM Pandinus imperator toxin 1 (Pi1)11 nM Kbot1 15 nM Bunodosema granulifera toxin (BgK) 39 nM Maurotoxin(MTX) 150 nM alpha-Dendrotoxin (DTX) 250 nM Parabuthus toxin 3 (PbTX3)492 nM Parabuthus toxin 1 (PbTX1) 800 nM ViTX 2 μM kappa-Hefutoxin 1(kappa-HfTX1) 150 μM Opisthacanus madagascariensis toxin 400 μM (OmTX3)

One of skill in the art will appreciate that because ion channels of thevarious families and subfamilies described herein share functional,sequence and structural similarity, the channel blockers shown in Table3 can also exhibit inhibitory effects on other ion channels in additionto Kv1.3. For example, Table 4 provide a list of selected channelblockers and their 1050 with One of skill in the art will readily beable to determine the 1050 of a particular channel blocker and an ionchannel using methods known in the art.

TABLE 4 IC50 values for various toxins Toxin Kv1.1 Kv1.2 Kv1.3 Kv1.5KCa3.1 Others Margatoxin 144 pM 520 pM to 110 pM to No effect No effect(MgTX) to 10 nM 675 pM 230 pM Kaliolotoxin 41 nM >1 μM 650 pM (KTX)Hongotoxin 31 pM 170 pM 86 pM No effect (HgTX1) Noxiustoxin >25 nM 2 nM1 nM >25 nM No effect (NTX) Heterometrus 7 nM No effect 12 pM 625 nMspinnifer toxin 1 (HsTX1) Maurotoxin No effect 110 pM 150 nM 1 nM (MTX)Orthochirus 600 pM 5.4 nM 14 pM >1 μM 225 nM scrobiculosus toxin (OSK1)OSK1- 400 pM 2.96 nM 3 pM >1 μM 228 nM Lys16Asp20 Pandinus 50 pMimperator toxin 2 (Pi2) Anuroctoxin 5 nM 730 pM No effect alpha- 1.1 nMto 17 nM 200 nM >1 μM Dendrotoxin 20 μM (DTX) Bunodosema 6 nM 15 nM to39 pM to 172 nM granulifera 25 nM 10 nM toxin (BgK) ViTX 2 μM No effect2 μM Correolide 21 nM to 10 nM to 90 nM to 7 nM to Kv1.6 19 nM to 430 nM700 nM 110 nM 1.1 μM 450 nM Kv1.4 10 nM PAC 200 nM to 200 nM to 149 nM200 nM to Kv1.6 200 nM to 400 nM 400 nM 400 nM 400 nM Psora-4 62 nM 49nM 3 nM 8 nM >5 μM Kv1.4 202 nM Kv1.7 100 nM PAP-1 65 nM 250 nM 2 nM 45nM 10 μM Kv1.6 62 nM Kv1.7 98 nM LiK-78282 22 μM 2.9 μM 280 nM 70 nM >30μM Kv1.4 170 nM Kv1.6 31 μM Kv3.2 127 μM CP-339818 62 μM 14 μM 230 nM 19μM >500 μM Kv1.4 300 nM Nav 10 nM Chalcone-16 1.2 μM >50 μM 400 nM 5.1μM >100 μM Kv1.7 10 μM Khellinone 3.1 μM 2 μM 280 nM 1.1 μM >100 μMdimer-2

Because different ion channels exhibit differential sensitivity to ionchannel toxins, one of skill in the art will readily be capable ofmodifying toxin insensitive ion channels by introducing one of moreamino acid substitutions into the amino acid sequence of the insensitiveion channel to render the ion channel sensitive to a toxin. Withoutwishing to be bound by theory, sensitivity of an ion channel to a toxincan depend on the sequence of the S5-S6 linker of the ion channel. Assuch, one of skill in the art will be capable of modifying the S5-S6region of an ion channel to render an ion channel sensitive to a toxinby introducing one or more mutations that mimic the S5-S6 sequence of atoxin sensitive channel. For example, it is known that the VGPCs KV 1.3and Kv2.1 differ significantly in their sensitivity to the scorpiontoxin AgTx2 (Garcia et al, Biochemistry 33, 6834-3839 (1994)). WhileKv1.3 is sensitive to various AgTx2 toxin isoforms, Kv2.1 is insensitiveto AgTx2.

Table 5 provides IC50 values between various pore blocking agents andgating modifier toxins and Kv1.1, Kv1.2, Kv1.3, Kv1.5, Kv2.1, Kv2.2 andKv2.2 having T359S, K360G, A366D, S367A, I383M, Y384T, and K386Vmutations.

TABLE 5 IC50 values between various pore blocking agents and toxins PoreBlocking Agent Gating Modifier Toxins Channel AgTx-1 Agtx-2 ChTx ShKHaTx GxTx-IE Shaker 0.16 nM  0.64 nM 227 nM — Weak — Kv1.1  136 nM 0.044nM 1500 nM 0.025 nM to >500 nM — 0.118 nM Kv1.2 — No/little 14 nM >1000nM >2000 nM No effect effect Kv1.3  1.7 nM 0.004 nM 0.19 nM 0.0009 nMto >500 nM No effect 0.011 nM Kv2.1 >2000 nM  >2000 nM  >2000 nM Noeffect 107 nM   2 nM Kv2.2 — — — — — 2.6 nM Kv2.1 — 0.007 nM — — 102 nM~2.0 nM  T359S K360G A366D S367A I383M Y384T K386V

One of skill in the art will appreciate that because replacement of theS5-S6 region in Kv2.1 with the S5-S6 region of Kv1.3 will yield amodified Kv2.1 protein that is sensitive to AgTx2 (Gross et al., Neuron,V. 13, 961-966 (1994). Thus in certain embodiments, the methodsdescribed herein can performed with an ion channel protein modified torender the ion channel sensitive to a scorpion toxin (e.g. AgTx2).

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Shaker protein comprising a modificationwherein the region comprising amino acid residues 424 to 451 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a F425G substitution, a T449H substitution or anycombination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Nav1.1 protein comprising a modificationwherein the region comprising amino acid residues 1422 to 1490 isreplaced with the sequence of amino acids 426 to 453 of Kv1.3. In oneembodiment, an ion channel modified to render the ion channel sensitiveto a scorpion toxin (e.g. AgTx2) suitable for use with the methodsdescribed herein comprises a Y1422S substitution, a A1429G substitution,a M1435D substitution, a D1436A substitution, a K1490V substitution orany combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Nav1.2 protein comprising a modificationwherein the region comprising amino acid residues 1422 to 1480 isreplaced with the sequence of amino acids 426 to 453 of Kv1.3. In oneembodiment, an ion channel modified to render the ion channel sensitiveto a scorpion toxin (e.g. AgTx2) suitable for use with the methodsdescribed herein comprises a Y1422S substitution, a A1419G substitution,a M1435D substitution, a D1426A substitution, a Y1450H substitution, aK1480V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Nav1.3 protein comprising a modificationwherein the region comprising amino acid residues 1407 to 1475 isreplaced with the sequence of amino acids 426 to 453 of Kv1.3. In oneembodiment, an ion channel modified to render the ion channel sensitiveto a scorpion toxin (e.g. AgTx2) suitable for use with the methodsdescribed herein comprises a Y1407S substitution, a A1414G substitution,a M1420D substitution, a D1421A substitution, a Y1445H substitution, aK1475V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Nav1.4 protein comprising a modificationwherein the region comprising amino acid residues 1234 to 1302 isreplaced with the sequence of amino acids 426 to 453 of Kv1.3. In oneembodiment, an ion channel modified to render the ion channel sensitiveto a scorpion toxin (e.g. AgTx2) suitable for use with the methodsdescribed herein comprises a Y1234S substitution, a A1241G substitution,a M1247D substitution, a D1248A substitution, a Y1272H substitution, aK1302V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Nav1.5 protein comprising a modificationwherein the region comprising amino acid residues 1409 to 1477 isreplaced with the sequence of amino acids 426 to 453 of Kv1.3. In oneembodiment, an ion channel modified to render the ion channel sensitiveto a scorpion toxin (e.g. AgTx2) suitable for use with the methodsdescribed herein comprises a Y1409S substitution, a A1416G substitution,a M1422D substitution, a D1423A substitution, a Y1447H substitution, aK1477V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Nav1.6 protein comprising a modificationwherein the region comprising amino acid residues 1403 to 1471 isreplaced with the sequence of amino acids 426 to 453 of Kv1.3. In oneembodiment, an ion channel modified to render the ion channel sensitiveto a scorpion toxin (e.g. AgTx2) suitable for use with the methodsdescribed herein comprises a Y1403S substitution, a A1410G substitution,a M1416D substitution, a D1417A substitution, a Y1441H substitution, aK1471V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Nav1.7 protein comprising a modificationwherein the region comprising amino acid residues 1385 to 1453 isreplaced with the sequence of amino acids 426 to 453 of Kv1.3. In oneembodiment, an ion channel modified to render the ion channel sensitiveto a scorpion toxin (e.g. AgTx2) suitable for use with the methodsdescribed herein comprises a Y1385S substitution, a A1392G substitution,a T1398D substitution, a I1399A substitution, a Y1423H substitution, aK1453V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Nav1.8 protein comprising a modificationwherein the region comprising amino acid residues 1357 to 1435 isreplaced with the sequence of amino acids 426 to 453 of Kv1.3. In oneembodiment, an ion channel modified to render the ion channel sensitiveto a scorpion toxin (e.g. AgTx2) suitable for use with the methodsdescribed herein comprises a Y1357S substitution, a A1364G substitution,a M1370D substitution, a D1371A substitution, a Y1395H substitution, aK1435V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Nav1.9 protein comprising a modificationwherein the region comprising amino acid residues 1247 to 1335 isreplaced with the sequence of amino acids 426 to 453 of Kv1.3. In oneembodiment, an ion channel modified to render the ion channel sensitiveto a scorpion toxin (e.g. AgTx2) suitable for use with the methodsdescribed herein comprises a Y1247S substitution, a A1254G substitution,a M1260D substitution, a D1261A substitution, a G1284M substitution, aY1285H substitution, a Q1335V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Cav1.1 protein comprising a modificationwherein the region comprising amino acid residues 1005 to 1112 isreplaced with the sequence of amino acids 426 to 453 of Kv1.3. In oneembodiment, an ion channel modified to render the ion channel sensitiveto a scorpion toxin (e.g. AgTx2) suitable for use with the methodsdescribed herein comprises a M1005S substitution, a L1019G substitution,a S1026A substitution, a C1088M substitution, a V1089H substitution, aQ1112V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Cav1.2 protein comprising a modificationwherein the region comprising amino acid residues 1077 to 1184 isreplaced with the sequence of amino acids 426 to 453 of Kv1.3. In oneembodiment, an ion channel modified to render the ion channel sensitiveto a scorpion toxin (e.g. AgTx2) suitable for use with the methodsdescribed herein comprises a M1077S substitution, a L1091G substitution,a S1098A substitution, a C1160M substitution, a V1161H substitution, aW1184V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Cav1.3 protein comprising a modificationwherein the region comprising amino acid residues 1092 to 1199 isreplaced with the sequence of amino acids 426 to 453 of Kv1.3. In oneembodiment, an ion channel modified to render the ion channel sensitiveto a scorpion toxin (e.g. AgTx2) suitable for use with the methodsdescribed herein comprises a M1092S substitution, a L1106G substitution,a S1113A substitution, a C1175M substitution, a V1176H substitution, aW1199V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Cav1.4 protein comprising a modificationwherein the region comprising amino acid residues 1066 to 1173 isreplaced with the sequence of amino acids 426 to 453 of Kv1.3. In oneembodiment, an ion channel modified to render the ion channel sensitiveto a scorpion toxin (e.g. AgTx2) suitable for use with the methodsdescribed herein comprises a M1066S substitution, a L1080G substitution,a C1149M substitution, a V1150H substitution, a W1173V or anycombination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Cav2.1 protein comprising a modificationwherein the region comprising amino acid residues 1454 to 1563 isreplaced with the sequence of amino acids 426 to 453 of Kv1.3. In oneembodiment, an ion channel modified to render the ion channel sensitiveto a scorpion toxin (e.g. AgTx2) suitable for use with the methodsdescribed herein comprises a L1454S substitution, a V1468G substitution,a C1537M substitution, a I1538H substitution, a W1563V substitution orany combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Cav2.2 protein comprising a modificationwherein the region comprising amino acid residues 1356 to 1465 isreplaced with the sequence of amino acids 426 to 453 of Kv1.3. In oneembodiment, an ion channel modified to render the ion channel sensitiveto a scorpion toxin (e.g. AgTx2) suitable for use with the methodsdescribed herein comprises a L1356S substitution, a V1370G substitution,a C1439M substitution, a I1440H substitution, a W1465V substitution orany combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Cav2.3 protein comprising a modificationwherein the region comprising amino acid residues 1344 to 1453 isreplaced with the sequence of amino acids 426 to 453 of Kv1.3. In oneembodiment, an ion channel modified to render the ion channel sensitiveto a scorpion toxin (e.g. AgTx2) suitable for use with the methodsdescribed herein comprises a L1344S substitution, a V1358G substitution,a V1365A substitution, a C1427M substitution, a I1428H substitution, aW1453V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Cav3.1 protein comprising a modificationwherein the region comprising amino acid residues 1193 to 1326 isreplaced with the sequence of amino acids 426 to 453 of Kv1.3. In oneembodiment, an ion channel modified to render the ion channel sensitiveto a scorpion toxin (e.g. AgTx2) suitable for use with the methodsdescribed herein comprises a A1193S substitution, a N1200G substitution,a G1206D substitution, a R1207A substitution, a V1281M substitution, aV1282H substitution, a T1326V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Cav3.2 protein comprising a modificationwherein the region comprising amino acid residues 1221 to 1344 isreplaced with the sequence of amino acids 426 to 453 of Kv1.3. In oneembodiment, an ion channel modified to render the ion channel sensitiveto a scorpion toxin (e.g. AgTx2) suitable for use with the methodsdescribed herein comprises a V1221S substitution, a V1222G substitution,a F1228D substitution, a F1229A substitution, a V1299M substitution, aV1230H substitution, a M1344V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Cav3.3 protein comprising a modificationwherein the region comprising amino acid residues 1033 to 1185 isreplaced with the sequence of amino acids 426 to 453 of Kv1.3. In oneembodiment, an ion channel modified to render the ion channel sensitiveto a scorpion toxin (e.g. AgTx2) suitable for use with the methodsdescribed herein comprises a A1033S substitution, a A1046D substitution,a W1047S substitution, a V1140M substitution, a V1141H substitution, aT1185V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv1.1 protein comprising a modificationwherein the region comprising amino acid residues 354 to 381 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a H355G substitution, a Y379H substitution or anycombination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv1.2 protein comprising a modificationwherein the region comprising amino acid residues 355 to 383 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a Q356G substitution, a V381H substitution, a T383Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv1.4 protein comprising a modificationwherein the region comprising amino acid residues 506 to 533 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a T506S substitution, a H507G substitution, a K531Hsubstitution, a 1533V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv1.5 protein comprising a modificationwherein the region comprising amino acid residues 462 to 489 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a T462S substitution, a H463G substitution, a R487Hsubstitution, a 1489V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv1.6 protein comprising a modificationwherein the region comprising amino acid residues 404 to 431 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a L405 substitution, a Y429H substitution, a M431Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv1.7 protein comprising a modificationwherein the region comprising amino acid residues 340 to 367 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a H341G substitution, a E347D substitution, a S348Asubstitution, a A365H substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv1.8 protein comprising a modificationwherein the region comprising amino acid residues 403 to 430 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a H404G substitution, a G411A substitution, a C428Hsubstitution, a T430V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv2.1 protein comprising a modificationwherein the region comprising amino acid residues 359 to 386 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a T359S substitution, a K360G substitution, a A366Dsubstitution, a S367A substitution, a 1383M substitution, a Y384Hsubstitution, a K386V or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv2.2 protein comprising a modificationwherein the region comprising amino acid residues 363 to 390 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a T363S substitution, a K364G substitution, a A370Dsubstitution, a S371A substitution, a 1387M substitution, a Y388Hsubstitution, a K390V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv3.1 protein comprising a modificationwherein the region comprising amino acid residues 382 to 409 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a T382S substitution, a H383G substitution, a 1389Dsubstitution, a G390A substitution, a Y407H substitution, a Q409Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv3.2 protein comprising a modificationwherein the region comprising amino acid residues 419 to 446 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a T419S substitution, a Q420G substitution, a 1426Dsubstitution, a G427A substitution, a Y444H substitution, a Q446Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv3.3 protein comprising a modificationwherein the region comprising amino acid residues 485 to 512 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a T485S substitution, a Y486G substitution, a 1492Dsubstitution, a G493A substitution, a Y510H substitution, a K512Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv3.4 protein comprising a modificationwherein the region comprising amino acid residues 418 to 445 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a T418S substitution, a D419G substitution, a 1425Dsubstitution, a G426A substitution, a Y443H substitution, a K445Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv4.1 protein comprising a modificationwherein the region comprising amino acid residues 354 to 381 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a T354S substitution, a N355G substitution, a A361Dsubstitution, a V379H substitution, a S381V substitution or anycombination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv4.2 protein comprising a modificationwherein the region comprising amino acid residues 352 to 379 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a K353G substitution, a A359D substitution, a V377Hsubstitution, a K379V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv4.3 protein comprising a modificationwherein the region comprising amino acid residues 349 to 376 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a K350G substitution, a A356D substitution, a S357Asubstitution, a V374H substitution, a K376V substitution or anycombination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv5.1 protein comprising a modificationwherein the region comprising amino acid residues 352 to 379 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a T352S substitution, a L353G substitution, a Q359Dsubstitution, a S360A substitution, a 1376M substitution, a Y377Hsubstitution, a K379V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv6.1 protein comprising a modificationwherein the region comprising amino acid residues 406 to 433 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a P406S substitution, a E407G substitution, a A413Dsubstitution, a C414A substitution, a V431H substitution, a R433Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv6.2 protein comprising a modificationwherein the region comprising amino acid residues 351 to 378 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a R351S substitution, a D352G substitution, a A358Dsubstitution, a S359A substitution, a V376H substitution, a R378Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv6.3 protein comprising a modificationwherein the region comprising amino acid residues 350 to 380 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a L350S substitution, a T351G substitution, a T358Dsubstitution, a S359A substitution, a Y380H substitution or anycombination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv6.4 protein comprising a modificationwherein the region comprising amino acid residues 400 to 427 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a L400S substitution, a E401G substitution, a A407Dsubstitution, a S408A substitution, a V425H substitution, a R427Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv7.1 protein comprising a modificationwherein the region comprising amino acid residues 403 to 454 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a H403S substitution, a K411G substitution, a Y417Dsubstitution, a V418A substitution, a C445M substitution, a D446Hsubstitution, a D454V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv7.2 protein comprising a modificationwherein the region comprising amino acid residues 460 to 535 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a A469S substitution, a S461G substitution, a R489Dsubstitution, a S490A substitution, a E530M substitution, a D531Hsubstitution, a G535V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv7.3 protein comprising a modificationwherein the region comprising amino acid residues 482 to 539 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a A482S substitution, a S487G substitution, a G493Dsubstitution, a D494A substitution, a K532M substitution, a K533Hsubstitution, a R539V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv7.4 protein comprising a modificationwherein the region comprising amino acid residues 431 to 461 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a R441D substitution, a P459M substitution, a T460Hsubstitution, a M461V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv7.5 protein comprising a modificationwherein the region comprising amino acid residues 485 to 542 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a K485S substitution, a V487G substitution, a A493Dsubstitution, a L494A substitution, a R535M substitution, a K536Hsubstitution, a R542V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv8.1 protein comprising a modificationwherein the region comprising amino acid residues 374 to 401 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a T374S substitution, a T375G substitution, a C381Dsubstitution, a 1398M substitution, a F399H substitution, a D401Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv8.2 protein comprising a modificationwherein the region comprising amino acid residues 439 to 466 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a T439S substitution, a N440G substitution, a H446Dsubstitution, a S447A substitution, a Y464H substitution, a E466Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv9.1 protein comprising a modificationwherein the region comprising amino acid residues 405 to 431 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a F405S substitution, a A410D substitution, a C411Asubstitution, a V427M substitution, a V428H substitution or anycombination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv9.2 protein comprising a modificationwherein the region comprising amino acid residues 358 to 384 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a L358S substitution, a A363D substitution, a C364Asubstitution, a V380M substitution, a V381H substitution, a G384Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv9.3 protein comprising a modificationwherein the region comprising amino acid residues 358 to 384 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a L358S substitution, a A363D substitution, a C364Asubstitution, a V380M substitution, a V381H substitution, a G384Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv10.1 protein comprising a modificationwherein the region comprising amino acid residues 501 to 561 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a I501S substitution, a Y506G substitution, a Y512Dsubstitution, a H513A substitution, a K554M substitution, a V555Hsubstitution, a K561V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv10.2 protein comprising a modificationwherein the region comprising amino acid residues 470 to 530 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a I470S substitution, a Y475G substitution, a Y481Dsubstitution, a H482A substitution, a K523M substitution, a V524Hsubstitution, a K530V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv11.1 protein comprising a modificationwherein the region comprising amino acid residues 662 to 722 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a I1662S substitution, a S668G substitution, a H674Dsubstitution, a T675A substitution, a A715M substitution, a V716Hsubstitution, a E722V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv11.2 protein comprising a modificationwherein the region comprising amino acid residues 514 to 574 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a 1514S substitution, a S520G substitution, a H526Dsubstitution, a T527A substitution, a A567M substitution, a V568Hsubstitution, a E574V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv11.3 protein comprising a modificationwherein the region comprising amino acid residues 665 to 725 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a I1665S substitution, a S671G substitution, a H677Dsubstitution, a M678A substitution, a V719H substitution, a E725Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv12.1 protein comprising a modificationwherein the region comprising amino acid residues 472 to 532 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a I1472S substitution, a Y477G substitution, a Y483Dsubstitution, a H484A substitution, a E525M substitution, a L526Hsubstitution, a D532V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv12.2 protein comprising a modificationwherein the region comprising amino acid residues 503 to 563 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a I503S substitution, a Y508G substitution, a Y514Dsubstitution, a H515A substitution, a E556M substitution, a L557Hsubstitution, a D563V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a Kv12.3 protein comprising a modificationwherein the region comprising amino acid residues 477 to 533 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a I477S substitution, a Y482G substitution, a Y488Dsubstitution, a H489A substitution, a E530M substitution, a L531Hsubstitution, a D533V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a HCN1 protein comprising a modificationwherein the region comprising amino acid residues 504 to 543 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a R504S substitution, a M512D substitution, a Y513Asubstitution, a S536M substitution, a Y537H substitution, a L543Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a HCN2 protein comprising a modificationwherein the region comprising amino acid residues 573 to 612 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a R573S substitution, a M581D substitution, a Y582Asubstitution, a S605M substitution, a Y606H substitution, a L612Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a HCN3 protein comprising a modificationwherein the region comprising amino acid residues 457 to 496 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a R457S substitution, a M465D substitution, a Y466Asubstitution, a S489M substitution, a Y490H substitution, a L496Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a HCN4 protein comprising a modificationwherein the region comprising amino acid residues 624 to 663 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a R624S substitution, a M632D substitution, a Y633Asubstitution, a S656M substitution, a Y657H substitution, a L663Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a CatSper1 protein comprising a modificationwherein the region comprising amino acid residues 393 to 424 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a T393S substitution, a S387G substitution, a W394Asubstitution, a S418M substitution, a T419H substitution, a W424Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a CatSper2 protein comprising a modificationwherein the region comprising amino acid residues 399 to 431 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a R399S substitution, a S401G substitution, a V407Dsubstitution, a S408A substitution, a S426M substitution, a K427Hsubstitution, a T431V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a CatSper3 protein comprising a modificationwherein the region comprising amino acid residues 297 to 329 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a I297S substitution, a H310D substitution, a I311Asubstitution, a E325M substitution, a N326H substitution, a K329Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a CatSper4 protein comprising a modificationwherein the region comprising amino acid residues 299 to 336 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a I299S substitution, a 1304D substitution, a V305Asubstitution, a E333M substitution, a E334H substitution or anycombination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is an Hv1 protein comprising a modificationwherein the region comprising amino acid residues 218 to 248 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a I218S substitution, a R223D substitution, a S224Asubstitution, a A240M substitution, a K241H substitution, a S248Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a KCa1.1 protein comprising a modificationwherein the region comprising amino acid residues 723 to 785 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a N723S substitution, a N726G substitution, a M732Dsubstitution, a R733A substitution, a F761M substitution, a S785Vsubstitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a KCa4.1 protein comprising a modificationwherein the region comprising amino acid residues 723 to 807 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a L723S substitution, a D730G substitution, a V736Dsubstitution, a T737A substitution, a S789M substitution, a Y790Hsubstitution, a A807V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a KCa4.2 protein comprising a modificationwherein the region comprising amino acid residues 642 to 725 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a Q642S substitution, a D649G substitution, a T655Dsubstitution, a T656A substitution, a Y707M substitution, a Y708Hsubstitution, a A725V substitution or any combination thereof.

In one embodiment, an ion channel modified to render the ion channelsensitive to a scorpion toxin (e.g. AgTx2) suitable for use with themethods described herein is a TPC1 protein comprising a modificationwherein the region comprising amino acid residues 659 to 696 is replacedwith the sequence of amino acids 426 to 453 of Kv1.3. In one embodiment,an ion channel modified to render the ion channel sensitive to ascorpion toxin (e.g. AgTx2) suitable for use with the methods describedherein comprises a E659S substitution, a C669G substitution, a V675Dsubstitution, a V691M substitution, a V692H substitution, a Y696Vsubstitution or any combination thereof.

Voltage-Sensitive Phosphatases

The methods described herein can also be used to identify compounds thatmodulate the activity of voltage-sensitive phosphatases (VSPs). VSPs arefour-transmembrane proteins that contain the characteristic voltagesensor domain of an ion channel, but instead of controlling an ion pore,the voltage sensor is linked to a cytoplasmic phosphoinositidephosphatase (Murata et al., 2005). One of skill in the art willappreciate that the amino acid substitutions described herein forinducing omega or sigma leaks can also be used to generate VSPs havingaltered voltage sensitivity. The prototypical member of the VSP familyis the voltage-sensor containing phosphatase (called CiVSP) isolatedfrom Ciona intestinalis (sea squirt; see Murata et al. (2005)Phosphoinositide phosphatase activity coupled to an intrinsic voltagesensor. Nature 435(7046):1239-1243). This enzyme consists of anintracellular phosphatase segment the activity of which is controlled bymembrane voltage acting via a VSD with strong sequence homology to thoseVSDs found in VGSC, VGCC, and VGPC alpha subunits; the VSD in CiVSPfamily members appears to act as a monomeric unit. (see, e.g., Okamuraet al. (2009) Voltage-sensing phosphatase: actions and potentials. JPhysiol. 587(Pt 3):513-520). Orthologues of CiVSP are found in humans,rodents, birds, and fishes (Okamura & Dixon (2011) Voltage-sensingphosphatase: its molecular relationship with PTEN. Physiology(Bethesda). 26(1):6-13). At least one human orthologue exists,phosphatidylinositol-3,4,5-triphosphate 3-phosphatase TPTE2, http://wwwuniprot.org/uniprot/Q6XPS3.

A multiple alignment of various VSPs and human Kv2.1 is provided in FIG.31. One of skill in the art will be capable of generating a VSP havingaltered voltage sensitivity by replacing one of more gating arginines,lysines or histidines in the S4 helix of the VSP with a substituentresidue having an uncharged polar side chain or with an amino acidresidue not having a positively charged side chain.

In one embodiment, an VSP having an altered voltage sensitivity suitablefor use with the methods described herein will be a CiVSP proteincomprising an amino acid substitution of one or more S4 helix gatingcharge amino acid residues at any of positions R223, R226, R229 or R232wherein the substitution replaces the positively charged arginineresidue with an amino acid substituent residue having an uncharged polarside chain or with an amino acid residue not having a positively chargedside chain. In one embodiment, the amino acid substitute at the gatingcharge position is a serine or an asparagine.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aCiVSP protein comprising a substitution of the tyrosine at position 160with an amino acid that is less hydrophobic than tyrosine wherein thesubstituted protein has faster deactivation kinetics relative toactivation or a substitution of the tyrosine at position 160 with anamino acid that is more hydrophobic than tyrosine wherein thesubstituted protein has slower deactivation kinetics relative toactivation.

In one embodiment, an VSP having an altered voltage sensitivity suitablefor use with the methods described herein will be a DrVSP proteincomprising an amino acid substitution of one or more S4 helix gatingcharge amino acid residues at any of positions R159, R162, R168 or R171,wherein the substitution replaces the positively charged arginineresidue with an amino acid substituent residue having an uncharged polarside chain or with an amino acid residue not having a positively chargedside chain. In one embodiment, the amino acid substitute at the gatingcharge position is a serine or an asparagine.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aDrVSP protein comprising a substitution of the phenylalanine at position100 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 100 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an VSP having an altered voltage sensitivity suitablefor use with the methods described herein will be a TPIP proteincomprising an amino acid substitution of one or more S4 helix gatingcharge amino acid residues at any of positions R100, R103, R109 or H112,wherein the substitution replaces the positively charged arginine orhistidine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aTPIP protein comprising a substitution of the phenylalanine at position81 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 81 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

In one embodiment, an VSP having an altered voltage sensitivity suitablefor use with the methods described herein will be a TPET2 proteincomprising an amino acid substitution of one or more S4 helix gatingcharge amino acid residues at any of positions R177, R180, R186 or H189,wherein the substitution replaces the positively charged arginine orhistidine residue with an amino acid substituent residue having anuncharged polar side chain or with an amino acid residue not having apositively charged side chain. In one embodiment, the amino acidsubstitute at the gating charge position is a serine or an asparagine.

In one embodiment, an ion channel having an S2 gating charge transportmutation suitable for use with the methods described herein will be aTPET2 protein comprising a substitution of the phenylalanine at position110 with an amino acid that is less hydrophobic than phenylalaninewherein the substituted protein has faster deactivation kineticsrelative to activation or a substitution of the phenylalanine atposition 110 with an amino acid that is more hydrophobic thanphenylalanine wherein the substituted protein has slower deactivationkinetics relative to activation.

Similarly, one of skill in the art will readily be able to design VSPmutants having altered voltage sensitivity on the basis of the L287C,F289C mutations in Kv2.1 (with or without MTSES or MTSET) or the C236S,V292C, and R294G mutations in Kv2.1 described herein.

Variants

The VGPs described herein can also comprise one or more non-conservativesubstitutions. In certain aspects, the methods described herein relateto methods for monitoring the activity of variant VGP comprising one ormore substituted amino acids and wherein the substituted amino acid isan amino acid having: (a) a similar side chain group, or (b) a similarside chain configuration, or (c) evolutionary positive relatedness, or(d) evolutionary neutral relatedness, or a nucleic acid encoding thesame. Thus, as used herein, the term VGP can also refer to any variantVGP described herein, including, but not limited to variants comprisingone or more amino acid substitutions with an amino acid having a similarside chain group, an amino acid having a similar side chainconfiguration, an amino acid having an evolutionary positiverelatedness, or an amino acid having an evolutionary neutralrelatedness.

In one embodiment, a variant VGP comprises an amino acid sequence havingat least about 75%, 80%, 85%, 90%, 95%, 98%, 99% identity with an aminoacid sequence of Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7,Nav1.8, Nav1.9, Cav1.1, Cav1.2, Cav1.3, Cav1.4, Cav2.1, Cav2.2, Cav2.3,Cav3.1, Cav3.2, Cav3.3, Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6, Kv1.7,Kv1.8, Kv2.1, Kv2.2, Kv3.1, Kv3.2, Kv3.3, Kv3.4, Kv4.1, Kv4.2, Kv4.3,Kv5.1, Kv6.1, Kv6.2, Kv6.3, Kv6.4, Kv7.1, Kv7.2, Kv7.3, Kv7.4, Kv7.5,Kv8.1, Kv8.2, Kv9.1, Kv9.2, Kv9.3, Kv10.1, Kv10.2, Kv11.1, Kv11.2,Kv11.3, Kv12.1, Kv12.2, Kv12.3, HCN1, HCN2, HCN3, HCN4, CatSper1,CatSper2, CatSper3, CatSper4, Hv1, KCa1.1, KCa4.1, KCa4.2, TPC1,Drosophila Shaker, or any other VGIC. In one embodiment, a variant VGPcomprises an amino acid sequence having at least about 75%, 80%, 85%,90%, 95%, 98%, 99% identity with an amino acid sequence of a VSP or aVGP described herein or known in the art.

As used herein, “sequence identity” means the percentage of identicalnucleotide or amino acid residues at corresponding positions in two ormore sequences when the sequences are aligned to maximize sequencematching, i.e., taking into account gaps and insertions. The comparisonof sequences and determination of percent identity between two sequencescan be accomplished using a mathematical algorithm. Techniques fordetermining sequence identity are well known to one skilled in the art,and include, for example, analysis with a sequence comparison algorithmor FASTA version 3.0t78 using default parameters (Pearson and Lipman,Proc Natl Acad Sci USA. 1988 April; 85(8):2444-8). In anothernon-limiting example, scoring of amino acid can be calculated using thePAM250 matrix as described in Dayhoff et al., (1978) in Atlas of ProteinSequence and Structure, ed. Dayhoff, M. (Natl. Biomed. Res. Found.,Silver Spring, Md.), Vol. 5, Suppl. 3, pp. 345-352.

Percent identity or percent similarity of a polypeptide sequence can bedetermined, for example, by comparing sequence information using the GAPcomputer program. The GAP program utilizes the alignment method ofNeedleman et al., 1970, as revised by Smith et al., 1981. Briefly, theGAP program defines similarity as the number of aligned symbols (i.e.,nucleotides or amino acids) that are similar, divided by the totalnumber of symbols in the shorter of the two sequences. The preferredparameters for the GAP program are the default parameters, which do notimpose a penalty for end gaps. See e.g., Schwartz et al., 1979; Gribskovet al., 1986. Nucleic acids that differ due to degeneracy of the geneticcode, and still encode the VGICs or variant VGICs, described herein areencompassed by the present disclosure.

VGPs falling within the scope of this invention can be produced by anynumber of methods, including but not limited to, error-prone PCR,shuffling, oligonucleotide-directed mutagenesis, assembly PCR, PCRmutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, and any combination thereof.

A variant VGP can comprise a conservative amino acid substitution inwhich an amino acid residue is replaced with an amino acid substituentresidue having a similar side chain group. VGPs falling within the scopeof this invention, can, in general, be accomplished by selectingsubstitutions that do not differ significantly in their effect onmaintaining (a) the structure of the polypeptide backbone in the area ofthe substitution, (b) the charge or hydrophobicity of the molecule atthe target site, or (c) the bulk of the side chain.

The VGPs described herein can be labeled, e.g., with a fluorophore orother detectable moiety, and/or fused to a polypeptide such as GFP, RFP,BFP and YFP. Suitable labels include but are not limited to a nucleicacid molecule, i.e., DNA or RNA, e.g., an oligonucleotide, a protein,e.g., a luminescent protein, a polypeptide, for instance, an epitoperecognized by a ligand, for instance, maltose and maltose bindingprotein, biotin and avidin or streptavidin and a His tag and a metal,such as cobalt, zinc, nickel or copper, a hapten, e.g., molecules usefulto enhance immunogenicity such as keyhole limpet hemacyanin (KLH),cleavable labels, for instance, photocleavable biotin, a fluorophore, achromophore, and the like.

VGPs fused to a fluorescent proteins (or non-fluorescent chromoproteins)can be readily generated by methods known in the art. Such fluorescentfusion proteins (or non-fluorescent chromoproteins) can be used todetect protein interaction by several methods, including but not limitedto immunoprecipitation and fluorescence resonance energy transfer(FRET). A fluorescent protein (or non-fluorescent chromoprotein) can bespecifically linked to the amino- or carboxyl-terminus of a VGP sequenceusing well known chemical methods, see, e.g., Chemical Approaches toProtein Engineering, in Protein Engineering: A Practical Approach (Eds.Rees et al., Oxford University Press, 1992). A fluorescent protein (ornon-fluorescent chromoprotein) can also be specifically insertedin-frame within a VGIC using well known chemical methods.

Fluorescent proteins (or non-fluorescent chromoproteins) useful inaspects of the invention include, e.g., those which have beengenetically engineered for superior performance such as, withoutlimitation, altered excitation or emission wavelengths; enhancedbrightness, pH resistance, stability or speed of fluorescent proteinformation; photoactivation; or reduced oligomerization orphotobleaching, see, e.g., Brendan P. Cormack et al., FACS—optimizedMutants of the Green Fluorescent Protein (GFP), U.S. Pat. No. 5,804,387(Sep. 8, 1998); Roger Y. Tsien & Roger Heim, Modified Green FluorescentProteins, U.S. Pat. No. 6,800,733 (Oct. 5, 2004); Roger Y. Tsien et al.,Long Wavelength Engineered Fluorescent Proteins, U.S. Pat. No. 6,780,975(Aug. 24, 2004); and Roger Y. Tsien et al., Fluorescent Protein SensorsFor Measuring the pH of a Biological Sample, U.S. Pat. No. 6,627,449(Sep. 30, 2003).

In one embodiment, the VGPs described herein can also be coupled with aradioisotope or enzymatic label to facilitate their detection. Forexample, the VGPs described herein can be isotopically-labeled where oneor more atoms are replaced or substituted by an atom having an atomicmass or mass number different from the atomic mass or mass numbertypically found in nature (i.e., naturally occurring). Suitableradionuclides that may be incorporated in compounds of the presentinvention include but are not limited to ²H (also written as D fordeuterium), ³H (also written as T for tritium), ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N,¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F, ³⁵S, ³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ¹²³I, ¹²⁴I, ¹²⁵Iand ¹³¹I. The radionuclide that is incorporated in the instantradio-labeled compounds can depend on the specific application of thatradio-labeled compound.

Alternatively, the VGPs described herein can be enzymatically labeledwith, for example, horseradish peroxidase, alkaline phosphatase, orluciferase, and the enzymatic label detected by determination ofconversion of an appropriate substrate to product.

In another embodiment, the VGPs described herein can be labeled with afluorescent dye, spin label, heavy metal or radio-labeled peptides.

In another embodiment, the VGPs described herein can comprise anon-natural amino acid. As used herein, a non-natural amino acid can be,but is not limited to, an amino acid comprising a moiety where achemical moiety is attached, such as an aldehyde- or keto-derivatizedamino acid, or a non-natural amino acid that includes a chemical moiety.A non-natural amino acid can also be an amino acid comprising a moietywhere a saccharide moiety can be attached, or an amino acid thatincludes a saccharide moiety. Examples of non-classical amino acidssuitable for use with the methods and compositions described hereininclude, but are not limited to, D-isomers of the common amino acids,2,4-diaminobutyric acid, alpha-amino isobutyric acid, 4-aminobutyricacid, Abu, 2-amino butyric acid, gamma-Abu, epsilon-Ahx, 6-aminohexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid,ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline,homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, beta-alanine, fluoro-amino acids,designer amino acids such as beta-methyl amino acids, C alpha-methylamino acids, N alpha-methyl amino acids, and amino acid analogs ingeneral.

The VGPs described herein can also comprise one or more amino acidanalog substitutions, e.g., unnatural amino acids such as alphaalpha-disubstituted amino acids, N-alkyl amino acids, lactic acid, andthe like. These analogs include phosphoserine, phosphothreonine,phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid,octahydroindole-2-carboxylic acid, statine,1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine,ornithine, citruline, .alpha.-methyl-alanine,para-benzoyl-phenylalanine, phenylglycine, propargylglycine, sarcosine,.epsilon.-N,N,N-trimethyllysine, .epsilon.-N-acetyllysine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,.omega.-N-methylarginine, and other similar amino acids and imino acidsand tert-butylglycine.

The VGPs described herein can further comprise polypeptide analogs, suchas peptide mimetics (Fauchere J, Adv. Drug Res. 15:29 (1986); Veber D Fand Freidinger R M, Trends Neurosci. 8:392-96 (1985); Evans B E et al.,J. Med. Chem 30:1229-39 (1987)). Generally, peptidomimetics arestructurally similar to a template polypeptide (i.e., a polypeptide thathas a biological or pharmacological activity), such as the VGPsdescribed herein, but have one or more peptide linkages replaced by alinkage selected from the group consisting of: —CH.sub.2NH—,—CH.sub.2S—, —CH.sub.2-CH.sub.2-, —CH.dbd.CH— (cis and trans),—COCH.sub.2-, —CH(OH)CH.sub.2-, and —CH.sub.2SO—, by methods known inthe art and further described in the following references: Spatola A Fin “Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins,”B. Weinstein, ed., Marcel Dekker, New York, p. 267 (1983); Spatola, A F,Vega Data (March 1983), Vol. 1, Issue 3, “Peptide BackboneModifications”; Morley J S, Trends Pharmcol. Sci. 1:463-68 (1980);Hudson D et al., Int. J. Pept. Prot. Res. 14:177-85 (1979)(—CH.sub.2NH—, CH.sub.2CH.sub.2-); Spatola A F et al., Life Sci.38:1243-49 (1986) (—CH.sub.2-S); Hann M M, J. Chem. Soc. Perkin Trans.1, 307-314 (1982) (—CH—CH—, cis and trans); Almquist R G et al., J. Med.Chem. 23:1392-98 (1980) (—COCH.sub.2-); Jennings-White C et al.,Tetrahedron Lett. 23:2533-34 (1982) (—COCH.sub.2-); EP 0 045 665(—CH(OH)CH.sub.2-); Holladay M W et al., Tetrahedron Lett., 24:4401-04(1983) (—C(OH)CH.sub.2-); Hruby V J, Life Sci. 31:189-99 (1982)(—CH.sub.2-S—). One example of a non-peptide linkage is —CH.sub.2NH—.

Such polypeptide mimetics can have advantages over polypeptideembodiments, including, for example: more economical production, greaterchemical stability, enhanced pharmacological properties (half-life,absorption, potency, efficacy, etc.), altered specificity (e.g., abroad-spectrum of biological activities), reduced antigenicity, andothers. Labeling of peptidomimetics can involve covalent attachment ofone or more labels, directly or through a spacer (e.g., an amide group),to non-interfering position(s) on the peptidomimetic that are predictedby quantitative structure-activity data and/or molecular modeling. Suchnon-interfering positions can be positions that do not from directcontacts with the macromolecules(s) to which the peptidomimetic binds toproduce the therapeutic effect. Derivatization (e.g., labeling) ofpeptidomimetics can be done without substantially interfering with thedesired biological or pharmacological activity of the peptidomimetic.The ability of any peptidomimetics to polypeptides can be assayed forthe ability to bind 1,4,-benzothiazepine or derivatives thereof usingmethods know to those skilled in the art.

Chemically modified derivatives of the VGPs described herein can also beprepared. For example, amides of the VGPs described herein can beprepared by techniques well known in the art for converting a carboxylicacid group or precursor, to an amide. One method for amide formation atthe C-terminal carboxyl group is to cleave the polypeptide, or fusionthereof from a solid support with an appropriate amine, or to cleave inthe presence of an alcohol, yielding an ester, followed by aminolysiswith the desired amine.

N-acyl derivatives of an amino group of the VGPs described herein can beprepared by utilizing an N-acyl protected amino acid for the finalcondensation, or by acylating a protected or unprotected polypeptide, orfusion thereof. O-acyl derivatives can be prepared, for example, byacylation of a free hydroxy polypeptide or polypeptide resin. Eitheracylation can be carried out using standard acylating reagents such asacyl halides, anhydrides, acyl imidazoles, and the like. Both N- andO-acylation can be carried out together, if desired.

Formyl-methionine, pyroglutamine and trimethyl-alanine can besubstituted at the N-terminal residue of VGPs described herein. Otheramino-terminal modifications include aminooxypentane modifications.

Systematic substitution of one or more amino acids of the VGPs describedherein with a D-amino acid of the same type (e.g., D-lysine in place ofL-lysine) can be used to generate additional amino acid sequencevariants.

Expression Systems

Expression of VGPs described herein can be by any method known in theart and include both cell-based expression systems and cell-freeexpression systems. For example, polypeptide of this invention can beexpressed in bacterial cells, insect cells (e.g., using baculovirusexpression vectors), yeast cells, amphibian cells, or mammalian cells.Suitable host cells are well known to one skilled in the art. For othersuitable expression systems for both prokaryotic and eukaryotic cells,see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

The VGPs described herein can be expressed in any suitable cell lineincluding oocytes. Cell lines suitable for expressing the VGPs describedherein can be of mammalian, amphibian, eukaryotic, archeal, or bacterialorigin. Both transient and stable methods of expression can be used inconjunction with the methods described herein. Alternatively the VGPscan be incorporated into a planar lipid bilayer, a liposome, or afunctional equivalent thereof. The VSDs can also be expressed, orincorporated, either alone, or as part of either the complete or partialVGP from which the VSD was originally derived, or as a part of a fusionprotein with another (partial or complete) VGP, or as part of a fusionprotein with another, unrelated protein.

Numerous methods exist for producing a VGP for analysis according to themethods described herein. Such methods, include, but are not limited to,exogenous expression systems. For example, VGPs can be expressedexogenously in frog oocytes or cell lines (e.g., embryonic kidney cells)to examine the behavior of populations of VGPs expressed at higherlevels relative to endogenous VGPs. One of skill in the art will readilyunderstand that expression of a VGP in an exogenous system may notaccount for interacting accessory proteins, a cell environment thatdiffers from the cell environment in which the VGP is normallyexpressed, differences in protein turnover, expression level artifacts,and improper trafficking to subcellular compartments in an artificialsystem. Methods are also known in the art for expressing andcharacterizing VGP in whole organisms such as genetically engineeredmice, worms, flies, or zebrafish.

Cells suitable for use with the methods described herein can be any cellhaving a cell membrane. In certain embodiments, a cell system can beused to express VGP in a cell so as to monitor the activity of the VGPusing any of the methods described herein. Cells suitable for use withthe methods described herein can be primary cells, cultured cells,established cells, normal cells, transformed cells, tumor cells,infected cells, proliferating, terminally differentiated cells or anycombination thereof.

Suitable cells include, but are not limited to bacterial (Gram-positiveor Gram-negative), archeabacterial, eukaryotic, prokaryotic, fungal,insect, avian, reptilian, oocyte, fly, zebrafish, nematode, fish,amphibian, or mammalian cells. The certain embodiments, cells suitablefor use with the methods described herein include, but are not limitedto mammalian cell lines (e.g. COS, CHO, HeLa, NIH3T3, HEK 293-T andPC12), amphibian cells (e.g., Xenopus embryos and oocytes), insect cells(e.g. D. melanogaster cells), yeast cells (e.g., S. cerevisiae, S.pombe, or Pichia pastoris), and prokaryotic cells (e.g. E. coli). In oneembodiment, the cell can be a Xenopus oocyte. The cells suitable for usewith the methods described herein can be from any species, including,but not limited to, human cells, mouse cells, rat cells, dog cells, pigcells, or Chinese hamster ovary cells. Other examples of types of cellssuitable for use with the methods described herein, include, but are notlimited to include immune system cells (e.g., B-cells, T-cells),oocytes, red blood cells, white blood cells, neuronal cells, epithelial,glia, fibroblast, cancer cells, and immortalized cells. Examples ofneuronal cells suitable for use with the methods described hereininclude, but are not limited to, squid axon, cerebellar Purkinje cells,neocortical pyramidal cells, thalamic neurons, CA1 hippocampal pyramidalcells, striatal neurons and mammalian CNS axons.

Examples of cell lines suitable for use with the methods describedherein include, but are not limited to 293-T cells, 3T3 cells, 721cells, 9L cells, A2780 cells, A2780ADR cells, A2780cis cells, A172cells, A20 cells, A253 cells, A431 cells, A-549 cells, ALC cells, B16cells, B35 cells, BCP-1 cells, BEAS-2B cells, bEnd.3 cells, BHK-21cells, BR 293 cells, BxPC3 cells, C2C12 cells, C3H-10T1/2 cells, C6/36cells, Cal-27 cells, CHO cells, COR-L23 cells, COR-L23/CPR cells,COR-L23/5010 cells, COR-L23/R23 cells, COS-7 cells, COV-434 cells, CMLT1 cells, CMT cells, CT26 cells, D17 cells, DH82 cells, DU145 cells,DuCaP cells, EL4 cells, EM2 cells, EM3 cells, EMT6/AR1 cells,EMT6/AR10.0 cells, FM3 cells, H1299 cells, H69 cells, HB54 cells, HB55cells, HCA2 cells, HEK-293 cells, HeLa cells, Hepa1c1c7 cells, HL-60cells, HMEC cells, HT-29 cells, Jurkat cells, J558L cells, JY cells,K562 cells, Ku812 cells, KCL22 cells, KG1 cells, KYO1 cells, LNCapcells, Ma-Mel 1, 2, 3 . . . 48 cells, MC-38 cells, MCF-7 cells, MCF-10Acells, MDA-MB-231 cells, MDA-MB-468 cells, MDA-MB-435 cells, MDCK IIcells, MDCK II cells, MG63 cells, MOR/0.2R cells, MONO-MAC 6 cells, MRCScells, MTD-1A cells, MyEnd cells, NCI-H69/CPR cells, NCI-H69/LX10 cells,NCI-H69/LX20 cells, NCI-H69/LX4 cells, NIH-3T3 cells, NALM-1 cells,NW-145 cells, OPCN/OPCT cells, Peer cells, PNT-1A/PNT 2 cells, Rajicells, RBL cells, RenCa cells, RIN-5F cells, RMA/RMAS cells, Saos-2cells, Sf-9 cells, SiHa cells, SkBr3 cells, T2 cells, T-47D cells, T84cells, THP1 cells, U373 cells, U87 cells, U937 cells, VCaP cells, Verocells, WM39 cells, WT-49 cells, X63 cells, YAC-1 cells, and YAR cells.

In certain embodiments, the cells suitable for use with the methodsdescribed herein will be cells that are transiently or stablytransfected with a VGP. Expression of a VGP in a cell according to themethods described herein can be driven by any method known in the art,including the use of constitutive, tissue-specific, cell-specific orinducible promoter element(s), enhancer element(s) or both. VGP that canbe expressed in a cell in accordance with the methods described hereincan be any VGP described herein.

For recombinant expression of one or more of the VGP described herein,the nucleic acid containing all or a portion of the nucleotide sequenceencoding the VGP can be inserted into an appropriate expression vector,i.e., a vector that contains the necessary elements for thetranscription and translation of the inserted protein coding sequence.The necessary transcriptional and translational signals can also besupplied by the native promoter of the component protein gene, and/orflanking regions (Protein Expression. A Practical Approach (Higgins andHames eds., Oxford University Press, 1999); Fernandez and Hoeffler, GeneExpression Systems. Using Nature for the Art of Expression (AcademicPress, 1999); and Rai & Padh, Current Science 80(9):1121-1128, (2001).

The methods described herein can also be used to monitor the activity ofan ion channel in a non-cell material, such as for example, anartificial membrane, a liposome, a phospholipid bilayer, and the like.Methods for introducing VGPs into artificial membrane, liposomes andphospholipid bilayers are known in the art and can be used inconjunction with the methods described herein.

In certain embodiments, co-expression of an inward rectifier channel canbe used in conjunction with the methods described herein to controlintracellular ion concentrations thereby allowing manipulation ofmembrane potential and to drive fractional inactivation of a VGIC underinvestigation.

In certain embodiments, the methods described herein can comprisemeasuring changes in VGP conductance by expressing a test VGP in a cell,a cellular fraction, an artificial membrane. Methods for measuringchanges in VGP conductance can comprise steps of stimulating orinhibiting conduction through one or more pores of a the VGP and/orcorrelating changes in experimental conditions with changes intransmembrane conductance or membrane potential. In certain embodiments,measurements can be compared to conductance measurements obtained incontrol or blank measurements.

The VGP described herein can be tested in any type of membrane known inthe art, including, but not limited to, a membrane from an intact cells,a membrane from a cellular fraction or an artificial membrane. Examplesof membranes from intact cells suitable for use with the methodsdescribed herein include, but are not limited to, oocyte membranes orcell line membranes. Examples of membranes from cellular fractionssuitable for use with the methods described herein include, but are notlimited to, membranes from luminal organelles such as nuclei, ribosomes,mitochondria, endoplasmic reticulum, Golgi apparatus, vacuoles, synapticvesicles, and lysosomes. Examples of artificial membranes suitable foruse with the methods described herein include, but are not limited to,phospholipid micelles, liposomes, micro- and nanocapsules, andsemi-liquid films on supportive structures.

Screening Methods and Compound Libraries

In certain aspects, the invention relates to methods useful foridentifying compounds which are capable of modifying the ionpermeability of VGICs that exhibits an ion leak when the VGIC is in aclosed or inactive conformation. In certain aspects, the inventionrelates to methods useful for identifying compounds which are capable ofmodifying activity of a VSP. Generally, test compounds are selected ifthey can alter the activity of a VGP (e.g., inhibit or reduce an ionleak when a mutant VGICs are in a closed or inactive conformation to astate or condition or level comparable to a wild-type or normal VGIC)

Examples of such compounds include, but are not limited to, smallorganic molecules including pharmaceutically acceptable molecules.Examples of small molecules include, but are not limited to,polypeptides, peptidomimetics, amino acids, amino acid analogs, nucleicacids, nucleic acid analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e., including heteroorganic and organometalliccompounds) having a molecular weight of less than about 10,000 grams permole, salts, esters, and other pharmaceutically acceptable forms of suchcompounds. Examples of other compounds that can be tested in the methodsof this invention include polypeptides, antibodies, nucleic acids, andnucleic acid analogs, natural products and carbohydrates. In certainembodiments, compounds suitable for testing with the methods describedherein can be from a peptide library, a phase display library, or from alibrary of known and/or previously characterized compounds.

A compound can have a known chemical structure but not necessarily havea known function or biological activity. Compounds can also haveunidentified structures or be mixtures of unknown compounds, for examplefrom crude biological samples such as plant extracts. Large numbers ofcompounds can be randomly screened from chemical libraries, orcollections of purified chemical compounds or collections of crudeextracts from various sources. The chemical libraries can containcompounds that were chemically synthesized or purified from naturalproducts. Methods of introducing test compounds to cells are well knownin the art.

Those having ordinary skill in the art will appreciate that a diverseassortment of compound libraries can be prepared according toestablished procedures, and tested for their influence VGIC function.The test compounds can be obtained using any of the numerous approachesin combinatorial library methods known in the art (see Lam K S,Anticancer Drug Des. 12:145-67 (1997)). Such compound libraries are alsoavailable from commercial sources such as ComGenex (U.S. Headquarters,South San Francisco, Calif.), Maybridge (Cornwall, UK), and SPECS(Rijswijk, Netherlands), ArQule, Tripos/PanLabs, ChemDesign andPharmacopoeia. The compounds identified in the screening methods of thisinvention can be novel or can be novel analogs or derivatives of knowntherapeutic compounds.

Assays for detecting, isolating and characterizing protein complexes arewell known in the art (e.g., immunoassays, activity assays,mass-spectrometry) and can be used to determine whether one or morecompounds described herein bind to a VGP. Methods for screening for amolecule that binds a VGIC can be performed using cell-free andcell-based methods known in the art (e.g. in vitro methods, in vivomethods or ex vivo methods). For example, an isolated VGP or VGP complexcan be employed, or a cell can be contacted with the candidate moleculeand the complex can be isolated from such contacted cells and theisolated complex can be assayed for activity or component composition.Methods for screening can involve labeling the component proteins of thecomplex with, for example, radioligands, fluorescent ligands or enzymeligands. VGPs can be isolated by any technique known in the art,including but not restricted to, co-immunoprecipitation, immunoaffinitychromatography, size exclusion chromatography, and gradient densitycentrifugation.

The following examples illustrate the present invention, and are setforth to aid in the understanding of the invention, and should not beconstrued to limit in any way the scope of the invention as defined inthe claims which follow thereafter.

EXAMPLES Example 1: Subcloning of cDNA Constructs

cDNA plasmids encoding VGICs will be subcloned into appropriate vectorssuitable for the generation of stable cell pools and stable cell lines.In certain embodiments, the pIRESpuro3 vector will be used for high,uniform expression in antibiotic resistant, stable pools to supportelectrophysiological characterization of Kv2.1 wild-type and mutantchannels. The mutant VGICs used will include “wild-type” Kv2.1, Kv2.1R300S mutant, and Kv2.1 R294C/R300S double mutant. All Kv2.1 constructscontain a set of mutations (T355S, K356G, A362D, S363A, I379M, Y380T,and K382V) in the channel's “turret region” that enables blockade of thecentral pore current by turret-selective (and pore-blocking) toxins,e.g., agitoxins.

Example 2: Generation of Wild-Type and Mutant Kv2.1 Stable Cell Pools,and Initial Evaluation of Central Pore Currents

Antibiotic-resistant stable cell pools from each Kv2.1 cDNA will begenerated. Electrophysiological characterization of expression of eachcell pool (the channel's central pore current) will be performed onIonWorks. The central pore current of each pool will be measured andcharacterized to determine I/V, G/V, and selectivity.

In certain embodiments, multiple transfections will be employed toobtain sufficiently high expression levels. Validation of cDNA plasmidpreparations can be performed by other methods, including, but notlimited to, qPCR or oocyte expression studies.

A host parental cell line (CHO) will be evaluated for sensitivity toantibiotic selection and toxicity of cDNA constructs to determineoptimal conditions for transfections. CHO cells will be transfected withmultiple concentrations of cDNA encoding the VGICs described herein.Antibiotic-resistant stable cell pools will be isolated and expanded.

For characterization of central pore ionic currents via IonWorks,voltage-clamp recordings in the presence and absence of pore-blockingtoxins (e.g., AgTx1, AgTx2, HgTx1, and/or ShK will be performed. Theefficiency of toxin blocking will be determined with replicatedprotocols (e.g. minimum of 250 successful recordings; seal R>50 MΩ).These methods will demonstrate stable expression of each cDNA andcharacterize Kv2.1 central pore currents. The data will be used tocompare effectiveness of about 2-4 different pore-blocking toxins andwhether there is efficient blockade of Kv2.1 central pore currents(sub-nM affinity; sub-pS central pore current) by one or morepore-blocking toxins.

Example 3: Manual Electrophysiology and Pharmacological Evaluation ofCentral Pore and Gating Pore Currents

Stable pools from each Kv2.1 cDNA will be used to evaluate thebiophysical and pharmacological properties of wild-type and mutant Kv2.1central pore and gating pore (leak) currents.

Manual voltage-clamp electrophysiology will be used to measure I/V andG/V relationships and selectivity for central pore current. Gating porecurrent pharmacology will be determined as a function of therelationship between I/V and G/V as well as the selectivity in thepresence of central pore and gating modifying peptides.

These results will demonstrate Kv2.1 central pore I/V and G/Vrelationships and ion selectivity as well as Kv2.1 gating pore I/V andG/V relationships and gating pore ion selectivity. These results willalso be used to determine whether there is a response, cessation of, orreduction in leak current in R294C, R300S and R300S Kv2.1 mutantsexposed to cysteine-reactive reagents.

Example 4: IonWorks Automated Patch Clamp Electrophysiology andPharmacological Evaluation of Central Pore and Gating Pore Currents

Stable pools from each Kv2.1 cDNA will be used to evaluate thebiophysical and pharmacological properties of wild-type and mutant Kv2.1central pore and gating pore (leak) currents.

IonWorks automated patch clamp electrophysiology will be used to measureI/V and G/V relationships and selectivity for central pore current.Gating pore current pharmacology will be determined as a function of therelationship between I/V and G/V as well as the selectivity in thepresence of central pore and gating modifying peptides.

These results will demonstrate Kv2.1 central pore I/V and G/Vrelationships and ion selectivity as well as Kv2.1 gating pore I/V andG/V relationships and gating pore ion selectivity. These results willalso be used to determine whether there is a response, cessation of, orreduction in leak current in R294C, R300S and R300S Kv2.1 mutantsexposed to cysteine-reactive reagents.

Example 5: Additional Experiments on the R294C, R300S Mutant Kv2.1 CellPool, Along with Control Experiments on the R300S Mutant Kv2.1 Cell Pool

Blockade of gating pore currents will be measured in the presence andabsence of cationic and anionic, membrane-impermeable cysteine-reactivereagents that react from the extracellular side of a membrane.

Example 6: Treating Voltage Sensing Domain Channelopathies

Resting state-specific voltage sensing domain leak current blockers(e.g. small molecule or modified toxin) will be used to assay thefunction of VGICs that exhibit leak currents. The assay will be used toidentify compounds suitable for treated VGIC related channelopathiesincluding, but not limited to autoimmune diseases (using a Kv1.3 blockerthat targets the pore domain), and Treat type 2 diabetes (using a Kv2.1blocker that targets the pore or the voltage sensing domain).

The method will be validated by targeting the KV2.1 voltage sensingdomain. Validation will be performed by monitoring resting state leakcurrents in wild type Kv2.1 and Kv2.1 comprising S4 helix mutations inthe voltage sensing domain. Leak currents will be blocked by selectivelytargeting the resting state of the VGIC using cysteine-tetheredreagents, voltage sensing domain toxins and small molecule libraryscreening. These results will confirm that targeting and blocking thevoltage sensing domain of VGICs is a valid therapeutic approach for thetreatment of channelopathies.

The methods described herein can be performed on any VGIC. In certainembodiments, the methods will involve a step of introducing a leakinducing mutation into a VGIC (e.g. an S4 mutation). In certainembodiments, the methods will involve a step of blocking main pore ioniccurrent (e.g. tetrodotoxin for Nav channels). In certain embodiments,the methods will involve a step of demonstrate that voltage sensingdomain mutant exhibits a leak current. In certain embodiments, themethods will involve a step of screening compound library (smallmolecules or biologics) for compounds capable of modulating of leakcurrent in mutated VGICs that exhibit a leak current.

Example 7: Mechanism of Voltage Gating in K+ Channels

The mechanism of ion channel “voltage gating”—how channels open andclose in response to voltage changes—has been debated since Hodgkin andHuxley's seminal discovery that the crux of nerve conduction is ion flowacross cellular membranes. Using all-atom molecular dynamicssimulations, shown herein is how a voltage-gated K⁺ channel switchesbetween activated and deactivated states. On deactivation, porehydrophobic collapse rapidly halts ion flow. Subsequent voltage-sensingdomain (VSD) relaxation, including inward, 15-Å S4-helix motion,completes the transition. On activation, outward S4 motion tightens theVSD-pore linker, perturbing linker-S6-helix packing. Fluctuations allowwater, then K⁺, to re-enter the pore; linker-S6 repacking stabilizes theopen pore. The results described herein show a mechanistic model for theNa⁺/K⁺/Ca²⁺ voltage-gated channel superfamily that reconciles apparentlyconflicting experimental data.

Hodgkin and Huxley discovered that voltage-regulated ion flow underliesnerve conduction (1). Only decades later were voltage-sensing domains(VSDs) identified as controlling the activity of voltage-gated K+, Na+,and Ca2+ channels (2-4), shifting these proteins between activated anddeactivated states in response to changes in transmembrane voltage(5-7). Different mechanistic models have been proposed to describe howconserved arginine and lysine “gating charge” residues on a VSDtransmembrane helix (S4) couple with the electric field to gate ionchannel conduction (8-10). Some experiments suggest substantial S4motion during gating (11), others far less (12). Also unresolved hasbeen whether S4 moves through a largely static electric field, orwhether the VSD instead reshapes the field around S4 during gating. Evenless clear has been how S4 triggers the attached channel pore domain togate conduction. Finally, it has been unknown whether other motions,either subtle or large-scale, are involved in voltage gating (6, 13),largely because, unlike the open state, no crystal structure of a fullydeactivated, closed-state voltage-gated channel exists.

To study the voltage-gated transition at the atomic level, we subjectedthe open conformation of the KV1.2/KV2.1 “paddle chimera” (10, 14)voltage-gated K+ channel to molecular dynamics (MD) simulations at bothhyperpolarizing (V<0 mV) and depolarizing (V>0 mV) voltages overexperimentally determined channel-gating timescales. Our all-atom systemcomprises the channel, either with (“T1+”) or without (“T1−”) themodulatory, but functionally nonessential cytoplasmic T1 domain (15,16),in a symmetric, neutral phospholipid bilayer [omitting modulatory,negatively charged lipids (17)], hydrated with 0.5 M KCl (18). Thesimulations were performed using a special-purpose machine designed forhigh-speed MD simulations (19).

At depolarizing control voltages, the (T1⁻) channel exhibited, steadyoutward conduction through a fully hydrated pore cavity (FIG. 16a, c andinset); K+ current and H2O/K+ permeation ratio (˜1) broadly agree withexperiment (20) and pore domain—only simulations (21). No significantconformational changes or gating charge displacement occurred,indicating that the paddle chimera crystal structure (10) embodies afully activated, open state.

In marked contrast, at hyperpolarizing voltages the channel went from anopen, inwardly conducting state to a closed, non-conducting state—theresting state. The channel exhibited a transient inward “tail” current,but conduction halted upon water leaving the hydrophobic pore cavity(“dewetting”) and concurrent pore closure (“cavity collapse”) at −20 μs(FIG. 16b, d and inset); the observed dewetting, as also seen inpore-only simulations (21), explains the osmotic sensitivity of theoverall gating process (21, 22). Subsequent inward S4 translation andlateral loosening of the VSDs from the pore domain completed thetransition over ˜100-200 μs (FIG. 17). Overall, the gating transitionthus consists of the channel moving from a VSD-“up,” VSD-pore apposedconformation to a VSD-“down,” VSD-pore loosened conformation (FIG. 16).The T1⁺ resting state exhibited less VSD-pore separation than T1.

The activated state VSDs delay channel closure by preventing porehydrophobic collapse into the intrinsically more stable closed state(19, 21). The activated-state VSDs delay channel closure by preventingpore hydrophobic collapse into the intrinsically more stable closedstate (21, 23). The time at which pore closure occurred (determined bythe pore cavity hydration level) and the tail current persistence timeobserved here, both ˜20 μs, are in line with the 20 μs experimental tailcurrent time constant [temperature-corrected Shaker data (18, 24); seeFIG. 23]. This closure time is, however, ˜10-fold longer than what wasfound in pore domain—only simulations (21), indicative of a VSD-imposeddelay of the closure. A control simulation with a different water model(18) also exhibited dewetting, after ˜30 μs, with little gating chargetransfer prior to dewetting (FIG. 27). The ˜100-200 μs taken to completethe activated-to-resting transition—including full gating chargedisplacement and VSD relaxation (FIG. 17)—is also in line with theexperimental value of ˜300 μs observed for the slow off-gating component(25).

The S4 helix—bearing gating-charge residues R1 (neutral Gln in thechimera; “R1(Q)”), R2, R3, R4, and K5—is the main VSD moving part S4translated ˜15 Å overall across the membrane in sequential steps whilerotating ˜120°, moving in a groove formed by the largely stationaryS1-S3a helices (FIG. 17). S3b, while more mobile than S1-S3a, did nottranslate inward to a notable extent. R4—centrally located in theactivated state at the point of strongest transmembrane electric field(FIG. 17c , inset)—initiated gating-charge movement; Phe233, a centralhydrophobic residue, separated the VSD extracellular and intracellularhydrated lumens throughout. R3 and R2 moved in turn, and inward S4motion typically stopped when R1(Q) reached Phe233. These observationssupport a recent gating model that emphasizes sequential motion of S4arginine residues past Phe233 (26).

As the gating-charge residues filed past Phe233, their side chains facedthe VSD lumens, not the membrane hydrophobic core; transient saltbridges formed by these gating-charge residues with acidic residues onS1 and S2 and with lipid negatively charged phosphodiester groupsfacilitated the transition. Relative to the activated state, the restingstate had fewer intra-VSD salt bridges but more S4-phosphateinteractions (FIG. 25). The results described herein show thatVSD-phosphodiester group interactions are functionally critical, whereasanionic lipids are modulatory, perhaps by interacting with gatingcharges or the T1 domain (17, 27).

In line with experimental gating charge values of 12-14 e (28), thecomplete transition into the fully relaxed state—all four VSDs “down”with all the gating-charge residues (R2-R4) inward of Phe233—yielded acalculated gating charge of 13.3±0.4 e (FIG. 17c and FIG. 21). ThisVSD-gating charge was solely accounted for by the ˜15-Å translation ofthe S4 helices through an electric field, focused over ˜15 Å, that wasfound to be similar in activated and resting VSD conformations (FIG. 17c, inset). The results described herein show that limited motion of asingle VSD (S4) toward the resting state suffices to close the pore.Charge displacements tied to early S4 motion—typically ˜1-7 e with atleast one R4 inward of Phe233 (FIG. 17c )—preceded pore closure, yet thepore always closed before all four VSDs were fully down. Experimentally,it is known that ˜25% charge displacement suffices to close the channel(29).

Initial S4 inward motion disrupted the extracellular VSD-pore domaininterface, resulting in domain separation: ˜14 Å for T1 and ˜5 Å forT1⁺, measured as the R1(Q)-Ala351 distance parallel to the membraneplane. VSD-pore separation contributed to a whole-proteinroot-mean-squared deviation (RMSD) of >14 Å (C_(a) atoms; relative tothe X-ray structure) for the T1 resting state and ˜9 Å for that of T1⁺(FIG. 17d ); the linker between the T1 domain and the VSD—an additionalconstraint not present in T1⁻—serves to reduce T1⁺ VSD mobility withinthe membrane plane. By contrast, the tetrameric pore domain exhibited,relative to the open-state crystal structure, only a modest ˜3-Å RMSDincrease, due to pore closure at ˜20-30 μs. Translation of S4 alone, oras the main moving part of the S3b-S4 paddle, resulted in large, >10-ÅRMSDs for S4 relative to the mostly stationary S1-S3a helices (RMSD<3Å).

Additional simulations revealed the major steps of channel activation(simulations 9-13, FIG. 21). The first (re)activation step of theresting-to-activated transition was examined by subjecting the T1⁺resting state, obtained above (simulation 9), to depolarizing voltages.Experimentally, T1⁺ channels activate faster than T1 deletion mutantchannels (16), presumably due to restraint of VSD mobility by the T1domain.

Upon depolarization, helix S4 immediately moved ˜7 Å outward,transferring ˜50% of the total gating charge in ˜75 μs (FIG. 18a, b ).Initially, gating-charge transfer is fast because most salt bridgesbetween S4 and the rest of the VSD are disrupted in the resting state;as S4 moved outward, these salt bridges began transiently to re-form,leading to a gradual slowing of S4 motion and gating-charge transfer. AsS4 movement neared completion the VSDs re-approached the activated state(FIG. 18a , see also FIG. 17). Experimentally, gating-charge transfermust be complete, in all four subunits, for the pore to open (8, 28). Inline with these experimental observations, full S4 outward movement (andgating-charge transfer) in one or two VSDs was insufficient to open thepore in the simulations described herein (FIG. 21).

The second reactivation step—the final cooperative transition (6, 7, 13)to the open state—was examined by starting from a T1⁻ configuration inwhich all VSDs, save one, were “up” (S4 helices fully outward), but forwhich the pore remained fully or partially closed (The “partiallyclosed” pore cavity contains ˜20 water molecules; FIG. 21, simulations10-12. The fully closed state resembles that observed in Na_(v)Ab (32)).These simulations, at depolarizing voltages, led to a fully activatedand conducting state within a few tens of microseconds (FIG. 18c-h ).

Experiments have shown that a single, cooperative transition precedesconduction; this final transition, which contributes only ˜5% of totalgating charge, occurs after all VSDs have moved fully up (8). In thesimulations described herein, little or no additional gating-chargetransfer occurred after the final outward motion of the S4 helix broughtthe S4-S5 linker into a tense conformation (FIG. 21). This final motionperturbed the packing between the S4-S5 linker and the S6 helix (FIG.18f, g ), leading to packing fluctuations, as shown by the linker-S6interaction energies in FIG. 18f . This weakened and fluctuating packingenabled partial pore opening and rapid partial rehydration—watermolecules re-entered the pore cavity in less than one, μs—allowingsubsequent K⁺ entry and initial slow, outward conduction (FIG. 18c-e ).

The partially open, partially hydrated, and slowly conducting porepersisted for a few microseconds before reaching the fully conductingstate (FIG. 18c, e ). The presence of ions in the cavity, whichsubsequently were driven outward through the selectivity filter (SF) bythe depolarizing voltage, further increased pore rehydration (FIG. 18c,d ). As the hydrostatic pressure within the cavity thereby increased,the lower gate, at the pore “PVP” motif, fully opened, with the Leu331(S5) and Pro405 (S6) side-chains interchanging positions (21). Thisinterchange caused S6 to kink at the PVP motif, widening the cavity andallowing it to finally become fully hydrated (FIG. 18c ). Concurrentopening of the upper (hydrophobic) gate, at Ile402 (FIG. 18h ), enabledSF site S5 to become populated with K⁺, thus increasing the cavity ionoccupancy by one (FIG. 18d, e ). K⁺ presence in S5 allowed formation ofthe critical “knock-on” conduction mechanism intermediate, (S5,[S4,S2];FIG. 18e ; 21), causing the channel to assume a fully conducting state(20.7±2.7 pA; FIG. 18c, e ). Early conduction occasionally slowed asLeu331 and Pro405 transiently back-interchanged; after the finalLeu331/Pro405 interchange took place, however, S6 and the S4-S5 linkersettled into a closely packed configuration that stabilized the openpore, and thus the activated, conducting state (FIG. 18 c, f, g).

Interaction of the S4-S5 linker and helix S6 is central to gating. InK_(v)1.5, pairs of interacting linker and C-terminal S6 residues havebeen identified (33). Mapped onto K_(v)1.2, these functionally importantinteractions are between residues [Ile316, Thr320] (linker) and [Asn412,Phe413, Phe416, Tyr417] (S6). These pairs, and linker residue Ala323,all stand out in residue contact maps (FIG. 18g ), highlightinglinker-S6 interaction as central to activation. Substitution of theK_(v)2.1 linker and S6 C-terminus into K_(v)1.5, moreover, has beenshown to confer K_(v)2.1 activation and deactivation kinetics to thisK_(v)1.5 chimera, while preserving K_(v)1.5 voltage dependency (33).Thus, the results described herein show that the linker and S6C-terminus together ultimately govern pore gating, independent of theVSD structural machinery that moves up or down during voltage sensing,irrespective of the details of the VSD structural machinery that movesup or down during voltage sensing,

The results described herein show the mechanistic model for K⁺ channelvoltage-gating shown in FIG. 19. This model integrates analysis ofatomic-level observations of both the activated-to-resting-state gatingtransitions and the complementary steps of the reverseresting-to-activated-state gating transition presented above (FIG. 21).This model shows that voltage-gated K⁺ channel opening requires themotion of four independent membrane-bound charged “particles,” whilechannel closing requires the motion of only one.

Beginning with the activated state (FIG. 19, state 1), ion depletion,hydrophobic dewetting, and closure of the pore cavity, with concurrentearly gating-charge inward movement, halt ionic conduction. Subsequentfull VSD relaxation—inward S4 translation by ˜15 Å relative to a largelyrigid S1-S3a VSD core coupled with ˜120° S4 rotation (FIG. 17b ) thatkeeps the gating-charge residues pointed toward the VSD lumens, andlateral VSD-pore separation due to VSD rotation and translation relativeto the pore—permits the pore to remain closed (FIG. 19, state 4).Translation of S4 gating-charge residues accounts for the gating charge.Channel activation reverses these steps. The key difference is that allfour VSDs must be “up” before the closed pore can reopen; a fullyoutward S4 perturbs the S4-S5 linker/S6 packing, thereby allowing water,and hence ion, re-entry and subsequent conduction, stabilized bylinker/S6 repacking.

The gating mechanism and resting-state structure described hereinreconcile the many apparently conflicting measures of voltage-gated K⁺channel conformation (FIGS. 16, 17, and 18). Given natural sequencevariability in functionally critical regions (S3b-S4 paddle; and theinteracting S4-S5 linker and S6), certain details of gating likely varybetween channels, including between wild-type K_(v)1.2 and theK_(v)1.2/K_(v)2.1 chimera that we used in our simulations. Yet, the factthat the S3b-S4 paddle can be swapped between channels to yield chimerasthat retain voltage-gating function (35, 36) shows that the keymechanistic aspects are broadly shared across the voltage-gated ionchannel superfamily

The results described herein show that channel opening and closing areenergetically asymmetric processes. Since the intrinsically most stablestate of the pore in isolation is dewetted and closed (21, 23), due tothe hydrophobic lining of the pore cavity (21), S4 need not actively“push” the S4-S5 linker down to close the pore. Channel activation does,however, require depolarization-driven work—the forcing of S4 backacross the membrane—ultimately to ultimately “pull” the S4-S5 linkertight, which perturbs its interaction with S6, leading to pore opening.Only when all gating-charge residues and S4-S5 linkers are fully “up”does the closed pore become sufficiently destabilized that fluctuationsof the lower gate, through perturbed linker-S6 packing, allow partialand then—as the last conformational transition duringactivation—complete cavity rewetting (FIG. 19).

The S4-S5 linker is tense in the activated state but relaxed in theresting state, perhaps explaining conservation of linker length: ashorter linker would inhibit channel closing, as S4 could not translateinward far enough, and a longer linker would inhibit opening, as evenfull S4 outward translation could not lead to an effective pull on thepore through the linker.

The atomic-level determination of the resting state conformationdescribed herein can be useful in guiding the development of drugs totreat those human channelopathies associated with the resting state(37). VSDs normally are impermeable to ions, but certain inherited S4gating-charge mutations permit abnormal cation conduction throughresting state “omega pores” (38), leading to, for example, cardiaclong-QT syndromes, various paralyses, and migraine (37, 39-40). VSDresidues accessible from the intracellular and extracellular sides (41,42) and residues thought to line the omega pore (43) map well onto theresting-state conformation described herein (FIG. 20a ). Thisresting-state VSD should thus exhibit omega currents, once an S4gating-charge residue is appropriately mutated to a smaller, polar,uncharged residue (37, 44, 45). Such mutation of Shaker R1 to histidineor serine permits H⁺ or alkali cation and guanidinium currents (38, 46).

The R2Ser mutation (37) was introduced into the resting stateconformation [R2-R4 down, (19, 38)]. The mutant VSD exhibitedsignificant inward K⁺ current (no Cl⁻ current). This current arisesbecause apposition of Phe233 with the mutated residue, which lacks thelarge, positive guanidinium group of the gating-charge residues, leadsto increased hydration of the VSD hydrophobic constriction and therebypermits permeation of cations (FIG. 20b, c ). Depolarization halted thecurrent and transferred ˜2 e of gating charge (FIG. 23)

The transition into the resting state, as well as the conformation ofthe state itself, demonstrates that the VSD omega and gating-permeationpathways are one and the same. Mutation of gating-charge residuesenables pathological cation leaks through the VSD along the identicalpathway taken by the physiological gating-charge guanidinium groups. Wethus provide a structural explanation for hyperpolarization-induced (aswell as depolarization-induced) cationic leak currents associated withchannelopathies in certain human voltage-gated ion channels.

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Example 8: Supporting Material, Mechanism of Voltage Gating in K⁺Channels

Methods

Simulation Systems.

The pore and voltage-sensing domains (residues 148-421) of the fullyfunctional (1) K_(v)1.2/K_(v)2.1 chimera [PDB entry 2R9R, (2)] wereembedded in a palmitoyl oleolyl phosphatidylcholine (POPC) bilayersolvated in ˜0.5 M KCl; the functionally nonessential T1 domain (3, 4,5) and regulatory β-subunit, both of which were present in the chimeracrystal structure, were omitted. Residue protonation states correspondedto pH 7. System sizes ranged from ˜107,000 atoms (110×110×87 Å³) to˜150,000 atoms (125×125×87 Å³), scaling with bilayer size. Allsimulations were initiated with K⁺ ions and water molecules atalternating positions in the selectivity filter (SF). Additionalsimulations that included the T1 domain were performed under identicalconditions (membrane composition, ion concentration, residue protonationstates, and initial SF occupancy). The system size was 230,000 atoms(125×125×145 Å³).

Simulations.

Five all-atom molecular dynamics simulations (FIG. 21; simulations 4-8:150, 215, 211, 216, and 256 μs) of the activated-to-resting transitionwere performed at hyperpolarizing voltages (−750≤V≤−375 mV), as well asthree control simulations (1-3: 80, 33, and 14 μs) at depolarizingvoltages (1, 2: +750 mV; 3: +375 mV). Three additional depolarizingsimulations (9-11: +750 mV, 20-33 μs), without the T1 domain, of theresting-to-activated transition were performed. These three simulationswere initiated from fully dewetted (pore fully closed) or partiallydewetted (pore partially open) conformations; in each simulation thevoltage-sensing domains (VSDs) were in the activated (“up”)conformation. Two depolarizing simulations (12: +375 mV, 80 μs; 13: +500mV, 68 μs), with the T1 domain included, of the resting-to-activatedtransition were also performed. Both simulations were initiated from theresting state, with the fully dewetted (pore fully closed) conformation,and with all but one (12) or all (13) of the VSDs in the completelydeactivated (“down”) conformation. The aggregate simulation time was1,500 μs (FIG. 21).

All simulations used the CHARMM27 force field for protein, ions, andwater (6, 7); the C36 force field was used for lipids (8). Torsionalbackbone corrections were added to SF residues Gly376 and Gly378 toprevent SF degradation on a timescale of microseconds (9; the localφ-maximum at 100° was converted to a local minimum through an m=6 ordertorsional correction: U=Σ_(m)(−1)^(m-1)·[(1+cos m(φ−φ′))/m!]. Theside-chain charges of aspartate, glutamate and arginine residues (“DER”correction; FIG. 22) were adjusted to weaken the guanidinium acetateassociation constant; this correction weakened the 4.6 M⁻¹ associationconstant obtained with standard CHARMM27 parameters to 0.9 M⁻¹, more inline with the experimental value of 0.3-0.5 M⁻¹(10, 11). The “DER2”correction (FIG. 22) further weakened the calculated associationconstant to 0.4 M⁻¹, to even more closely reproduce the experimentalvalue.

Simulations were performed on a special-purpose machine, Anton (12),designed for molecular dynamics simulations (NPT ensemble; 310 K, 1 bar,Berendsen coupling scheme with one temperature group [13]). All bondlengths to hydrogen atoms were constrained using M-SHAKE (14). Van derWaals and short-range electrostatic interactions were cut off at 10 Å.Long-range electrostatic interactions were calculated using the GaussianSplit Ewald method (15) with a 64×64×64 FFT mesh. The simulation timestep was 2 fs (2.5 fs in simulations 8, 12, and 13); long-rangeelectrostatics were evaluated every third step. The protein wasinitially relaxed in the membrane, for >2 μs, without application of atransmembrane potential. Thereafter, a potential was imposed,implemented as a constant electric field of ±0.01-0.2kcal·mol⁻¹·Å⁻¹·e⁻¹, as described (9). The field was increased linearlyto full strength over 1 μs and held constant thereafter; for simulationsat hyperpolarizing voltage, after the transition to the resting statewas either fully or nearly complete (defined as a gating charge of ˜10e), the magnitude of the applied voltage was typically lowered by 50%;in simulation 8, however, the voltage was increased by 33%, and thesimulations were then continued for at least another 10 μs, to ensurethat the resting state remained stable. Trajectories were saved at300-ps intervals and were analyzed using HiMach (16), which integratesVMD and its plugins (17).

Simulations 2-7, 9, and 11 (FIG. 21) used the DER charges (FIG. 22),simulations 1 and 10 used standard CHARMM27 charges, and simulations 8,12, and 13 used DER2 charges. Simulation 7 was initiated with standardcharges, but was then restarted with DER charges. At first, thissimulation, at hyperpolarizing voltage, was extended for 55 μs beyondthe time of cavity dewetting (from 45 to 100 μs) without significantgating-charge displacement relative to depolarizing control simulations1 and 2. The switch to DER charges at 45 μs, however, permitted VSDrelaxation to commence. At depolarizing voltages, no differences werediscernible between simulations performed with standard versus DERcharges. To accommodate more complete VSD relaxation, additional lipidmolecules were added to simulations 6 and 7 (107,000 atoms increased to150,000 atoms), at 24 and 62 μs. Simulations 9-11 were started fromsnapshots of (hyperpolarizing) simulation 7 (with 107,000 atoms), at 45μs (simulation 9) and 48 μs (simulations 10 and 11), in which all VSDgating-charge residues were still “up” (outward of Phe233) and in whichthe pore cavity was either partially (simulation 9) or fully dewetted(simulations 10 and 11). Application of a reversed, depolarizing voltageled to complete cavity rewetting within 10 μs and concurrentsteady-state outward K⁺ conduction (19.1±0.1, 20.7±2.7, and 20.7±3.7 pAin simulations 9, 10, and 11, respectively; cf. simulation 1, 18.8±0.8pA).

Simulations that included the T1 domain (FIG. 21; simulations 8, 12, and13) were performed at reduced voltage magnitudes. Simulation 8 (−375 mV)was initiated with DER charges, but was then restarted with DER2charges, at 78 μs, because no noticeable gating charge displacement hadoccurred with DER charges at this relatively weak applied voltage;cavity dewetting was observed with DER charges, at 33 μs (FIG. 21). Theswitch to DER2 charges led to complete VSD relaxation, over ˜200 μs. Thelast ˜50 μs was performed with a slightly increased voltage magnitude(33%), to drive the activating-to-resting state transition tocompletion. Simulations 12 (+375 mV) and 13 (+500 mV) used DER2 chargesthroughout.

Experimental time constants were corrected for temperature differencesbetween simulation (T=310 K) and experiment (T′=293 K) using therelationship: τ_(T)=τ_(T′)Q₁₀ ^((T′−T)/10). For the tail current,τ_(293 K)=0.308 ms and Q₁₀˜4.8 (18); for the slow off-gating component,τ_(293 K)=3-4 ms and Q₁₀˜4 (19). Throughout, error bars and “±”represent standard error of the mean.

Analysis.

Trajectories, accessed from a parallel disk cache system called Zazen(60), were analyzed using HiMach (61), which integrates VMD and itsplugins (62).

Helix Rotation Calculations.

The local S4 helical axis was obtained from the principal inertial axesof gating-charge residues R2, R3, or R4 together with the two precedingand two following residues (N, C_(a), C, and O atoms only). Thatprincipal axis nearest the mean C═O direction of these five residues waschosen as the local helical axis (x-axis; “roll”), rotation around whichis shown in FIG. 17b . The local y-axis (“pitch”) was defined as themean of the vectors (perpendicular to the roll axis) through the C_(β)atoms of the preceding and following residues; the pitch axis thuscoincides roughly with the R2, R3, or R4 side chain. The local z-axis(“yaw”) was defined as z=x×y. Eulerian angles (rotations in order: yaw;pitch; roll) for this local reference frame were computed relative tothe simulation start (all angles zero). Yaw and pitch, which togetherdefine the (changing) direction of the S4 helix axis, variedlittle)(±20° throughout. Roll was mostly negative, i.e., viewed from theextracellular side, S4 rotated in the counter-clockwise directionrelative to the rest of the VSD as helix S4 moved inward (FIG. 17b ).

Transmembrane-Potential Calculations.

For both activated and resting states, the transmembrane-voltage profilethrough the VSD was computed from simulations of a single VSD (residues148-321) embedded in a single bilayer, imposing a depolarizing potentialdifference (V) implemented as a constant electric field (0 or +0.2kcal·mol⁻¹·Å⁻¹·e⁻¹ [0 or +750 mV]), as above.

The fractional potential drop, relative to V, was probed along themembrane normal (z) at the gating-charge residues R2-R6 (R1(Q) and R0were also included for the resting state) using free-energycalculations, performed with Desmond (20). First, eight charging(2={0.00, 0.15, 0.31, 0.45, 0.63, 0.77, 0.89, 1.00}) and then ninecoupling (2={0.000, 0.107, 0.175, 0.225, 0.282, 0.366, 0.501, 0.710,1.000}) windows were used. Each window was simulated for 2.1 ns, and theBennett acceptance method (21) was used to obtain the Gibbs free energy.The fractional potential drop, f(z), was obtained as (22):f(z)=−ΔΔG(z)/qV+(z+L_(z)/2)/L_(z). ΔΔG(V; z)=ΔG(V>0; z)−ΔG(V=0; z) isthe potential (free) energy difference, between introducing a gatingcharge (q) in the presence or absence of the electric field. Thenon-uniform charge distribution across the VSD modifies (nonlinearly)the linear potential drop, (z+L_(z)/2)/L_(z), contributed by theconstant electric field (truncated outside±L_(z)/2, noting that L_(z)=30Å fully encompasses the region over which the transmembrane field acts).

The values calculated above for the fractional potential drop were fitto f(z)=1/[exp(−c(z−z′)+1], with resulting parameters [c, z′] of [0.52Å⁻¹, 4.4 Å] and [0.44 Å⁻¹, 5.0 Å] for the activated (A) and resting (R)states. From these f(z) curves, 95% of the potential drop was found tobe distributed over an ˜15-Å-wide region in both states (FIG. 17c ,inset). The activated-state parameters were used in computation of thegating-charge displacement; see main text and FIG. 17c , inset. The(single VSD) gating charge can also be estimated as Q=Σ_(i)q_(i)[f(z_(i,R)))−f(z_(i,A))]=2.5 e, summing over gating-charge residuesR2-R6 only; use of this subset of S4 residues implies that this estimateis lower, by ˜1 e, than the value of ˜13.5 e expected for the either theentire S4 or the full VSD (FIG. 17c ).

“Omega” Pore Simulations.

For simulation of omega currents (FIG. 23), two resting-state VSDconfigurations obtained through simulations—one with R2 at Phe233 (R2-R4“down”; simulation 4 at t=72 μs) and the other with R0 at Phe233 (R0,R2-R4 “down”; simulation 3 at t=200 μs)—were converted to omega pores byintroduction of R2Ser or R0Asn mutations, respectively. Glu226 was alsomutated to aspartate, because the longer glutamate side chain couldblock or interfere with the omega current [it is known that thecorresponding Shaker mutation, Glu283Asp, enhances omega currents (23)];back-mutation of Asp226 to glutamate had no systematic effect on omegacurrents. The mutant VSDs were embedded in a hydrated POPC bilayer(system size 40,000 atoms), as described above (both CHARMM27 and C36parameters were used for the lipids, but no difference resulted), andwere simulated at hyperpolarizing voltages for ˜10 to ˜130 μs. Toterminate the omega currents and reactivate the sensor, two (inwardlyconducting) snapshots (both at 10 μs) from the simulations were takenand then imposed reverse (depolarizing) voltages (see FIG. 23). Theaggregate simulation time of the omega pore constructs was ˜300 μs.

SUPPORTING REFERENCES

-   1. X. Tao, R. MacKinnon, Functional analysis of Kv1.2 and paddle    chimera Kv channels in planar lipid bilayers. J. Mol. Biol. 382,    24-33 (2008).-   2. S. B. Long, X. Tao, E. B. Campbell, R. MacKinnon, Atomic    structure of a voltage-dependent K+ channel in a lipid membrane-like    environment. Nature 450, 376-382 (2007).-   3. A. M. VanDongen, G. C. Frech, J. A. Drewe, R. H. Joho, A. M.    Brown, Alteration and restoration of K+ channel function by    deletions at the N- and C-termini. Neuron 5, 433-443 (1990).-   4. H. T. Kurata, G. S. Soon, D. Fedida, Altered state dependence of    C-Type inactivation in the long and short forms of human Kv1.5. J.    Gen. Physiol. 118, 315-332 (2001).-   5. W. R. Kobertz, C. Miller, K+ channels lacking the    ‘tetramerization’ domain: implications for pore structure. Nature    Struct. Biol. 6, 1122-1125 (1999).-   6. A. D. MacKerell, Jr. et al., All-atom empirical potential for    molecular modeling and dynamics studies of proteins. J. Phys. Chem.    B 102, 3586-3616 (1998).-   7. A. D. MacKerell, Jr., M. Feig, C. L. Brooks, III, Extending the    treatment of backbone energetics in protein force fields:    Limitations of gas-phase quantum mechanics in reproducing protein    conformational distributions in molecular dynamics simulations. J.    Comput. Chem. 25, 1400-1415 (2004).-   8. J. B. Klauda et al., Update of the CHARMM all-atom additive force    fields for lipids: Validation on six lipid types. J. Phys. Chem. B    114, 7830-7843 (2010).-   9. M. Ø. Jensen et al., Principles of conduction and hydrophobic    gating in K+ channels. Proc. Natl. Acad. Sci. USA 107, 5833-5838    (2010).-   10. C. Tanford, The association of acetate with ammonium and    guanidinium ions. J. Am. Chem. Soc. 76, 945-946 (1954).-   11. B. Springs, P. Haake, Equilibrium constants for association of    guanidinium and ammonium ions with oxyanions: The effect of changing    basicity of the oxyanion. Bioorg. Chem. 6, 181-190 (1977).-   12. D. E. Shaw et al., Millisecond-scale molecular dynamics    simulations on Anton. Proc. Conf. High Performance Computing,    Networking, Storage and Analysis (SC09) (ACM Press, New York, 2009).-   13. H. J. C. Berendsen, J. P. M. Postma, W. F. van Gunsteren, A. Di    Nola, J. R. Haak, Molecular dynamics with coupling to an external    bath. J. Chem. Phys. 81, 3684-3690 (1984).-   14. V. Krautler, W. F. van Gunsteren, P. H. Hünenberger, A fast    SHAKE algorithm to solve distance constraint equations for small    molecules in molecular dynamics simulations. J. Comput. Chem. 22,    501-508 (2001).-   15. Y. Shan, J. L. Klepeis, M. P. Eastwood, R. O. Dror, D. E. Shaw,    Gaussian split Ewald: A fast Ewald mesh method for molecular    simulation. J. Chem. Phys. 122, 054101:1-13 (2005).-   16. T. Tu et al., A scalable parallel framework for analyzing    terascale molecular dynamics simulation trajectories. Proceedings of    the ACM/IEEE Conference on Supercomputing (SC08) (ACM Press, New    York, 2008).-   17. W. Humphrey, A. Dalke, K. Schulten, V M D: Visual Molecular    Dynamics. J. Mol. Graphics 14, 33-38 (1996).-   18. B. M. Rodriguez, F. Bezanilla, Transitions near the open state    in Shaker K+-channel: Probing with temperature. Neuropharmacology    35, 775-785 (1996).-   19. B. M. Rodriguez, D. Sigg, F. Bezanilla, Voltage gating of Shaker    K+ channels. The effect of temperature on ionic and gating    currents. J. Gen. Physiol. 112, 223-242 (1998).-   20. K. J. Bowers et al., Scalable algorithms for molecular dynamics    simulations on commodity clusters. Proc. ACM/IEEE Conf. on    Supercomputing (SC06) (ACM Press, New York, 2006).-   21. C. H. Bennett, Efficient estimation of free energy differences    from Monte Carlo data. J. Comput. Phys. 22, 245-268 (1976).-   22. B. Roux, The membrane potential and its representation by a    constant electric field in computer simulations. Biophys. J. 95,    4205-4216 (2008).-   23. F. Tombola, M. M. Pathak, E. Y. Isacoff, Voltage-sensing    arginines in a potassium channel permeate and occlude    cation-selective pores. Neuron 45, 379-388 (2005).

Example 9: Expression of Shaker Channel Constructs in Xenopus Oocytes

The coding cDNAs of Shaker “wild-type” (hereinafter WT) base construct(Shaker H4, with intact inactivation), Shaker R362S with alpha pore open(hereinafter R1 S) and Shaker with the alpha pore closed by mutationW434F (herein after R1 S/W434F) were cloned into a high expressionvector pBSTA. The C-terminus of all constructs was tagged with eGFP.Transcripted cRNA was produced using the mMessage mMachine kit (AmbionInc.).

Two to four days past injection of cRNA into stage V-VI Xenopus oocytes(NASCO), omega currents (Iω) and currents through the alpha pore (Iα)were recorded with the two electrode voltage-clamp technique making useof a TURBO TEC-05X amplifier (npi electronic GmbH). Currents werefiltered at 10 kHz and sampled at 20 kHz. The bath solution contained(in mM): NaCl 96, KCl 2, CaCl₂ 1, MgCl₂ 1 and HEPES 5 (pH 7.5).Voltage-recording and current-injecting microelectrodes were filled with3 M KCl and pulled to have resistances between 0.2 and 1 MΩ (see Storket al., 2007). The pClamp software package v.10.1 (Molecular Devices,Inc.) was used for data acquisition and analysis.

Example 10: Recording of Alpha Currents (Iα)

Pulses (50 ms) were applied from −60 to +60 mV in 10 mV steps at 1 Hzfrom a holding potential of either −80 mV (WT, R1S/W434F) or −50 mV(R1S) (see insets in FIGS. 33B and 33D respectively) to measure alphacurrents (Iα). Passive leak currents were subtracted online using a P/4procedure: pClamp software generates a series of scaled-down replicasweeps of the main stimulus waveform. These subsweeps are of the sameduration as the main sweep, but of lesser amplitude; amplitudes in thesubsweeps are inversely proportional to the number of subsweeps selected(usually 4, hence P/4). The cell's response to the subsweeps is used tocalculate the degree of passive cellular current leak. This is thensubtracted from acquired data on the associated input signal.

Example 11: Recording of Omega Currents (Iω)

Pulses (20 ms) from +60 to −300 mV were applied in 20 mV steps at 1 Hzfrom a holding potential of either −80 mV (WT, R1S/W434F) or −50 mV(R1S) (see FIG. 34B) to measure omega currents (Iω) without leaksubtraction.

Example 12: Drug Application and Analysis

Lanthanum(III) chloride heptahydrate (LaCl₃.7H₂O, Sigma-Aldrich) wasfreshly dissolved in the bath solution. La³⁺ was applied to Xenopusoocytes by means of a fast perfusion technique (ScreeningTool, npielectronic GmbH as described by Baburin et al., 2006). Origin softwarev.7.0 (OriginLab Corp.) was employed for data analysis. Inhibition ofomega currents (in %) was defined as (1−Iω,drug/Iω,control)*100, whereIω, drug is the current response in the presence of a givenconcentration of drug and Iω,control is the control omega current. Dataare given as mean±SE (n=number of experiments).

Example 13: Demonstration of the I/V Relationship for WT and R1S ShakerChannels with Intact Inactivation Expressed in Xenopus Oocytes

Electrophysiological characterization of WT and R1S Shaker channelsexpressed in Xenopus oocytes injected with the corresponding cRNAs areshown in FIG. 33. I/V relationships of WT (A), R1S (C) are shown.Currents were normalized to the maximal current at +60 mV (mean datafrom 7 oocytes (A) and 4 oocytes (C)). Normalized current amplitudes atgiven voltages are shown as mean values±S.E. (hidden by symbols).Typical potassium outward currents through WT (B) and mutant R1S (D)channels are illustrated on the right panel. The insets display thecorresponding voltage protocols and holding potentials.

Example 14: Demonstration that Inward Gating Pore Leak Currents at LargeNegative Voltages are not Present in WT Shaker with Intact Inactivation

Electrophysiological characterization of WT Shaker channels expressed inXenopus oocytes injected with the corresponding cRNA are shown in FIG.34. The I/V curve in (A) illustrates that Iω are not present in WTShaker. Test pulses were applied from +60 mV to −300 mV (illustrated inB). Currents were normalized to the maximal current at +60 mV (data from7 oocytes). Normalized current amplitudes at given voltages are shown asmean values±S.E. (hidden by symbols). Typical potassium outward currentsthrough WT (C) are illustrated on the lower panel. Same experiment athigher resolution is shown in (D) illustrating an unspecific inwardconductance developing at pulses negative to −260 mV (see also meanvalues in A at −280 and −300 mV). This current was also seen in noninjected oocytes and may represent instability of the membrane at verynegative voltages.

Example 15: Demonstration that Inward Gating Pore Leak Currents at LargeNegative Voltages are Present in R1S Shaker and that the Gating PoreLeak Current is Blocked by La³⁺ Ions

I/V curves in FIG. 35 (A) illustrate that Iω is present in R1S and thatthis current is blocked by La³⁺. Test pulses were applied from +60 mV to−300 mV (same voltage steps as in FIG. 34B applied from a holdingpotential of −50 mV). Currents were normalized to the maximal current at+60 mV. Control R1S I/V curve (squares) and I/V curves in the presenceof 30 (circles), 100 (triangles) and 300 .mu.M La³⁺ (diamonds) are shown(data from 4 oocytes). Normalized current amplitudes at given voltagesare shown as mean values±S.E. Typical potassium outward currents throughR1S and omega currents in control (B) and in the presence of 30 μM (C)and 300 μM La³⁺ (D) are illustrated.

Example 16: Demonstration that Inward Gating Pore Leak Currents at LargeNegative Voltages are Still Present in R1S Shaker when the Alpha Pore isClosed by the W434F Mutation

I/V curve in FIG. 36(A) illustrates that Iω is present in R1S/W434F.This construct conducts no alpha current (compare outward currentsduring depolarising pulses positive to 0 mV with FIGS. 33-35). Testpulses were applied from +60 mV to −300 mV (same voltage protocol as inFIG. 34 B). Current amplitudes at given voltages are shown as meanvalues±S.E (data from 6 oocytes). (B) and (C) illustrate the absence ofoutward currents and at a higher resolution (C) the presence of omegacurrents in construct R1S/W434F. (D) Iω was induced by voltage stepsfrom a holding potential of −80 mV to −200 mV (see inset) Inhibition ofIω induced by 100 μM La³⁺ occurred in a “use-dependent” manner. Thelower trace (control, in the absence of La³⁺) is superimposed by twentycurrents during 20 ms pulses applied at a frequency of 1 Hz. During thefirst pulse 100 μM La³⁺ blocked 31.8±3.6%. At steady state omegacurrents were typically blocked by 51.5±5.9% (data from 5 oocytes).

Example 17: R1S and R1S/W434F Shaker, but not WT Shaker, Exhibit aGating Pore Leak Current that can be Blocked by a Pharmacological Agent

The data presented in FIG. 33, FIG. 34, FIG. 35, and FIG. 36 togetherillustrate that WT and R1S display the expected IN curves in regards tooutward currents activated at voltages positive to −20 mV. Omegacurrents for R1S and R1S/W434F Shaker only started to activate athyperpolarizing pulses negative to −80 mV. These currents were onlyrecorded in R1S and R1S/W434F but not in WT Shaker. Omega currents wereblocked in a concentration-dependent manner by La³⁺. Omega currents areblocked by La³⁺ in a state-dependent manner (FIG. 36).

REFERENCES FOR EXAMPLES 9-17

-   Baburin I, Beyl S and Hering S (2006) Automated fast perfusion of    Xenopus oocytes for drug screening. Pflügers Arch 453(1): 117-23.-   Stork D, Timin E N, Berjukow S, Huber C, Hohaus A, Auer M, et    al. (2007) State dependent dissociation of hERG channel inhibitors.    Br J Pharmacol 151(8): 1368-76.-   Tombola F, Pathak M M, Gorostiza P, Isacoff E Y (2007) The twisted    ion-permeation pathway of a resting voltage-sensing domain. Nature    445(7127): 546-9.

Example 18: Expression of Kv2.1 Channel Constructs in Xenopus Oocytes

The coding cDNAs of KV2.1 “wild-type” (hereinafter WT) base construct(hKv2.1-m7-c3 is human Kv2.1, residues 1-858, mutated to confer AgTx2sensitivity and to remove extracellular cysteine residues) wassynthesized (Entelchon GmbH) and cloned into the high expression vectorpBSTA.

Mutations Kv2.1 R294N (hereinafter R0N), Kv2.1 R300S (hereinafter R2S),Kv2.1 R294C/R300S (hereinafter R0C/R2S), Kv2.1 R293S/R294S (hereinafterR-1S/R0S), Kv2.1 R293S/R294C (hereinafter R-1S/ROC), Kv2.1R293S/R294S/R300S (hereinafter R-1S/R0S/R2S), Kv2.1 R293S/R294C/R300S(hereinafter R-1S/R0C/R2S) were introduced using the QuikChange®Lightning Site-Directed Mutagenesis Kit (Stratagene) with mutagenicprimers according to the manufacturer's instructions. All constructswere checked by restriction site mapping and sequencing.

Transcripted cRNA was produced using the mMessage mMachine kit (AmbionInc.). Two to four days past injection of cRNA into stage V-VI Xenopusoocytes (NASCO) potassium currents through the alpha pore (Iα) wererecorded with the two electrode voltage-clamp technique making use of aTURBO TEC-05X amplifier (npi electronic GmbH). Currents were filtered at10 kHz and sampled at 20 kHz. The bath solution contained (in mM): NaCl96, KCl 2, CaCl2 1, MgCl2 1 and HEPES 5 (pH 7.5). Voltage-recording andcurrent-injecting microelectrodes were filled with 3 M KCl and pulled tohave resistances between 0.2 and 1 MΩ (see Stork et al., 2007). ThepClamp software package v.10.1 (Molecular Devices, Inc.) was used fordata acquisition and analysis.

Example 19: Voltage Protocol, Compounds, and Drug Application andAnalysis

For recording of alpha currents (Iα), 100 ms pulses were applied from−60 to +60 mV in 10 mV steps at 1 Hz from a holding potential of −80 mVto the Kv2.1 channel constructs to measure alpha currents (Iα). Passiveleak currents were subtracted online using a P/4 procedure: pClampsoftware generates a series of scaled-down replica sweeps of the mainstimulus waveform. These subsweeps are of the same duration as the mainsweep, but of lesser amplitude; amplitudes in the subsweeps areinversely proportional to the number of subsweeps selected (usually 4,hence P/4). The cell's response to the subsweeps is used to calculatethe degree of passive cellular current leak. This is then subtractedfrom acquired data on the associated input signal. There was no evidencefor omega currents in this voltage range that could potentiallyinterfere with the leak subtraction.

AgTx2 (Sigma Aldrich) was reconstituted according to manufacturer'sinstructions with 10 mM Tris, pH 7.5, containing 100 mM sodium chloride,0.1% bovine serum albumin, and 1 mM EDTA.

AgTx2 was applied to Xenopus oocytes by means of a fast perfusiontechnique (ScreeningTool as described by Baburin et al., 2006). Originsoftware v.7.0 (OriginLab Corp.) was employed for data analysis.Inhibition of alpha currents (in percentage) was defined as(1−Iα,drug/Iα,control)*100, where Iα,drug is the current response in thepresence of a given concentration of AgTx2 and Iα,control is the controlpotassium current. Data are given as mean±SE (n=number of experiments).

Example 20: Demonstration of the I/V Relationships, Outward PotassiumCurrents, and Mean Outward Currents for WT and Mutant Kv2.1 Channels

The I/V relationships of various Kv2.1 channels constructs expressed inXenopus oocytes injected with the corresponding cRNAs are shown in FIG.37. WT (A), R0N (B), R2S(C), R0C/R2S (D), R-1S/R0S (E), R-1S/R0C (F),R-1S/R0S/R2S (G) and R-1 S/R0C/R2S(H) Kv2.1 channel constructs expressedin Xenopus oocytes are illustrated. Currents were normalized to themaximal current at +60 mV (data from 9 (A), 7 (B), 6 (C), 4 (D), 8 (E),4 (F), 7 (G) and 4 oocytes (H)). Normalized current amplitudes at givenvoltages are shown as mean values±S.E. (hidden by symbols). Typicalpotassium outward currents though the designated channel constructs areshown in FIG. 38: WT (A), R0N (B), R2S(C), R0C/R2S (D), R-1S/R0S (E),R-1S/R0C (F), R-1S/R0S/R2S (G) and R-1S/R0C/R2S(H). Mean maximal outwardcurrents at +60 mV through the designated Kv2.1 constructs (in μA; twobatches, n≥3) are shown in FIG. 39.

Example 21: Demonstration that the Central Pore (Alpha) Current of Kv2.1WT and Mutant Channels is Blocked by AgTx2

UV relationships for Kv2.1 channels in the absence and presence of AgTx2are shown in FIG. 40. WT (A) and R2S (D) channels are shown in theabsence (control, squares) and presence of 1 nM AgTx2 (circles). Peakcurrents were normalized to the maximal current at +60 mV. Normalizedpeak current amplitudes at given voltages are shown as mean values±S.E.(hidden by symbols). The mean inhibition of the maximal outward currentat +60 mV amounted 66.5±2.9% (n=4, WT) and 67.8±2.8% (n=5, R2S). Typicalpotassium outward currents through WT and R2S Kv2.1 channels in control(B, E) and in the presence of 1 nM AgTx2 (C, F) are illustratedrespectively.

The data in FIG. 37, FIG. 38, FIG. 39, and FIG. 40 illustrate that WT,R0N, R2S, R0C/R2S, R-1S/R0S, R-1S/R0C, R-1S/R0S/R2S and R-1S/R0C/R2Sdisplay the expected I/V curves in regards to outward (alpha) currentsactivated at voltages positive to −60 mV. Outward current activated atvoltages positive to −40 mV (see Gross et al., 1994). Furthermore, bycloning the constructs into pBSTA we achieved high expression levels.This is evident from the mean maximal currents at +60 mV ranging from8.5±0.9 μA in R-1S/R0S/R2S to 42.0±5.2 μA in R0N (WT: 41.6±5.4 μA). WTand R2S alpha currents were blocked by 1 nM AgTx2 by 66.5±2.9% and67.8±2.8% respectively.

REFERENCES FOR EXAMPLES 18-21

-   Baburin I, Beyl S and Hering S (2006) Automated fast perfusion of    Xenopus oocytes for drug screening. Pflügers Arch 453(1): 117-23.-   Stork D, Timin E N, Berjukow S, Huber C, Hohaus A, Auer M, et    al. (2007) State dependent dissociation of hERG channel inhibitors.    Br J Pharmacol 151(8): 1368-76.-   Gross A., Abramson T. and MacKinnon R. (1994) Transfer of the    scorpion toxin receptor to an insensitive potassium channel. Neuron,    13, 961-966.

Example 22: Further Expression of Kv2.1 Channel Constructs in XenopusOocytes

The coding cDNAs of KV2.1 “wild-type” (hereinafter WT) base construct(hKv2.1-m7-c3 is human Kv2.1, residues 1-858, mutated to confer AgTx2sensitivity and to remove extracellular cysteine residues) wassynthesized (Entelchon GmbH) and cloned into the high expression vectorpBSTA.

Mutations Kv2.1 R294N (hereinafter R0N), Kv2.1 R300S (hereinafter R2S),Kv2.1 R294C/R300S (hereinafter R0C/R2S), Kv2.1 R293S/R294S (hereinafterR-1S/R0S), Kv2.1 R293S/R294C (hereinafter R-1S/ROC), Kv2.1R293S/R294S/R300S (hereinafter R-1S/R0S/R2S), Kv2.1 R293S/R294C/R300S(hereinafter R-1S/R0C/R2S) were introduced using the QuikChange®Lightning Site-Directed Mutagenesis Kit (Stratagene) with mutagenicprimers according to the manufacturer's instructions. All constructswere checked by restriction site mapping and sequencing.

Transcripted cRNA was produced using the mMessage mMachine kit (AmbionInc.). Two to four days past injection of cRNA into stage V-VI Xenopusoocytes (NASCO) potassium currents through the alpha pore (Iα) wererecorded with the two electrode voltage-clamp technique making use of aTURBO TEC-05X amplifier (npi electronic GmbH). Currents were filtered at10 kHz and sampled at 20 kHz. The bath solution contained (in mM): NaCl96, KCl 2, CaCl2 1, MgCl2 1 and HEPES 5 (pH 7.5). Voltage-recording andcurrent-injecting microelectrodes were filled with 3 M KCl and pulled tohave resistances between 0.2 and 1 MΩ (see Stork et al., 2007). ThepClamp software package v.10.1 (Molecular Devices, Inc.) was used fordata acquisition and analysis.

Example 23: Voltage Protocols, Compounds, and Drug Application andAnalysis

Recording of alpha currents (Iα): Pulses of 100 ms were applied from −60to +60 mV in 10 mV steps at 1 Hz from a holding potential of −80 mV tothe Kv2.1 channel constructs to measure alpha currents (Iα). Passiveleak currents were subtracted online using a P/4 procedure: pClampsoftware generates a series of scaled-down replica sweeps of the mainstimulus waveform. These subsweeps are of the same duration as the mainsweep, but of lesser amplitude; amplitudes in the subsweeps areinversely proportional to the number of subsweeps selected (usually 4,hence P/4). The cell's response to the subsweeps is used to calculatethe degree of passive cellular current leak. This is then subtractedfrom acquired data on the associated input signal. Recording of omegacurrents (Iω): Pulses of 10 ms were applied from a holding potential of−80 to −300 mV in 20 mV steps at 0.3 Hz to the Kv2.1 channel constructs.Iω were recorded without leak subtraction.

AgTx2 (Sigma Aldrich) was reconstituted according to manufacturer'sinstructions with 10 mM Tris, pH 7.5, containing 100 mM sodium chloride,0.1% bovine serum albumin, and 1 mM EDTA. LaCl3 was from Sigma Aldrich;MTSEA, MTSES and MTSET were from Biotium.

La³⁺, AgTx2, MTSEA, MTSES and MTSET and were applied to Xenopus oocytesby means of a fast perfusion technique (ScreeningTool as described byBaburin et al., 2006). Origin software v.7.0 (OriginLab Corp.) wasemployed for data analysis. Inhibition of inward currents was defined as1−Iω,drug/Iω,control, where Iω,drug is the current response in thepresence of a given concentration of lanthanum (La³⁺) and Iω, control isthe control current through the omega pore. The concentration-inhibitioncurve was fitted using the Hill equation:Iω,drug/Iω,control=A+A/(1+(C/IC50)^(nH)) where IC50 is the concentrationat which Iω inhibition is half-maximal, C is the applied La³⁺concentration, A is the fraction of Iω current that blocked and nH isthe Hill coefficient. Data are given as mean±SE (n=number ofexperiments).

Example 24: Demonstration that Inward Gating Pore Leak Currents (Iω) arePresent in Certain Mutant, but not in WT or Other Mutant, K_(v)2.1Channels at Large Negative Voltages

Significant inward omega currents (Iω) were induced by triple mutationsR-1S/R0C/R2S and R-1S/R0S/R2S, as illustrated in FIG. 41.Current-voltage relationships of WT, R0N, R2S, R0C/R2S, R-1S/R0C,R-1S/R0S, R-1S/R0C/R2S and R-1S/R0S/R2S Kv2.1 channel constructsnormalized to maximal outward current at +60 mV. Inward currents wererecorded during 10 ms hyperpolarising voltage steps from a holdingpotential of −80 mV to −300 mV (20 mV steps) in Xenopus oocytes injectedwith the corresponding cRNAs. Normalized current amplitudes at givenvoltages are shown as mean values±S.E. (n≥4, two batches of oocytes).Currents were not leak subtracted. Inward currents through the singleand double mutants R0N, R2S, R0C/R2S, R-1S/R0C, and R-1S/R0S were notsignificantly different from WT.

Example 25: Demonstration that the Inward Gating Pore Leak Current (Iω)is not Blocked by AgTx2

Inward currents at negative potentials in oocytes expressing the Kv2.1mutant R-1S/R0S/R2S were not inhibited by 10 nM AgTx2. This AgTx2concentration is 10 times higher than the 1 nM AgTx2 which inhibitsabout 70% of the potassium outward current in WT and R2S Kv2.1constructs. Illustrated in FIG. 42 are the inward current-voltagerelationships of Kv2.1 construct R-1S/R0S/R2S in the absence (control,squares) and after 5 minutes in the presence of 10 nM AgTx2 (filledcircles). Inward currents after 5 ms were normalized to the maximaloutward current at +60 mV. Normalized inward current amplitudes at givenvoltages are shown as mean values±S.E. (n≥4, two batches of oocytes).

Example 26: Demonstration that the Inward Gating Pore Leak Current (Iω)is Blocked by La³⁺ Ions

Inward currents at negative potentials in oocytes expressingR-1S/R0S/R2S were blocked by La³⁺ in a concentration-dependent manner,as illustrated in FIG. 43 and FIG. 44. FIG. 43 (A) illustrates theinward current-voltage relationships of construct R-1S/R0S/R2S in theabsence (control; squares) and in the presence of the indicatedconcentrations of La³⁺ (other symbols). Inward currents after 5 ms incontrol and La³⁺ were normalized to the maximal outward current at +60mV. Normalized inward current amplitudes at given voltages are shown asmean values±S.E. (n≥4, two batches of oocytes). FIGS. 43 (B, C)illustrate typical inward currents during hyperpolarising pulses throughR-1S/R0S/R2S in the absence (B) and in the presence of 10 mM La³⁺ (C).FIG. 44 (A) illustrates the concentration-dependent inhibition of theinward currents after 5 ms (normalized to control) through R-1S/R0S/R2Sat −300 mV. The concentration-inhibition curve was fitted to the Hillequation. FIG. 44 (B) illustrates superimposed inward currents at −300mV in the absence and in the presence of 30 μM, 100 μM, 300 μM, 1 mM, 3mM and 10 mM La³⁺. A half-maximal inhibition concentration (IC50) of785±419 μM was estimated (nH=0.8±0.1; n>4, two batches of oocytes).

Example 27: Demonstration that the Inward Gating Pore Leak Current (Iω)is not Blocked by Certain Reagents

Cysteine-reactive reagents MTSET, MTSES, or MTSEA were applied for 5minutes to oocytes expressing the Kv2.1 R-1S/R0C/R2S mutant channel. Toenable access of Cys294 during this period the channels were repeatedlyactivated by applying test pulses from −80 mV to 40 mV at a frequency of0.3 Hz. FIG. 45 illustrates the inward current-voltage relationships ofKv2.1 mutant R-1S/R0C/R2S in the absence of (squares) and after 5minutes application of 1 mM MTSEA (filled circles). FIG. 46 illustratesthe inward current-voltage relationships of Kv2.1 mutant R-1S/R0C/R2S inthe absence of (squares) and after 5 minutes application of 1 mM MTSES(filled circles). FIG. 47 illustrates the inward current-voltagerelationships of Kv2.1 mutant R-1S/R0C/R2S in control (squares) andafter 5 minutes application of 1 mM MTSET (filled circles). Inwardcurrents after 5 ms in the absence of reagent or in the presence of theMTSEA, MTSES, or MTSET reagents were normalized to the inward current at−300 mV in the absence of reagent. Normalized inward current amplitudesat the indicated voltages are shown as mean values±S.E. (n≥4, twobatches of oocytes).

Example 28: Suitability of Certain Channel Voltage Sensor Mutants forScreening

Only the Kv2.1 R-1 S/R0C/R2S and R-1S/R0S/R2S mutants conductsignificantly larger inward currents during hyperpolarising test pulsesthan the WT channel. There was no evidence for Iω in the other Kv2.1channel constructs. Our finding that Kv2.1 R0N, R2S, R0C/R2S mutants donot open an omega pore highlights structural differences in the voltagesensors between Shaker and Kv2.1. Shaker R1S mutant did exhibit an openomega pore. R-1 and RO are not present in Shaker and Kv2.1 has acysteine in the Shaker R0 position (see alignment illustrated in FIG.48). Taken together, the Shaker R1S and the Kv2.1 R-1S/R0S/R2S triplemutant are suitable for drug screening on Iω. High expression levels inoocytes and corresponding large current amplitudes are required forreliable Iω measurements. Other channel types that are relevant forchannelopathy mutations might also be considered for such studies.

REFERENCES FOR EXAMPLES 22-28

-   Baburin I, Beyl S and Hering S (2006) Automated fast perfusion of    Xenopus oocytes for drug screening. Pflügers Arch 453(1): 117-23.-   Stork D, Timin E N, Berjukow S, Huber C, Hohaus A, Auer M, et    al. (2007) State dependent dissociation of hERG channel inhibitors.    Br J Pharmacol 151(8): 1368-76.-   Khodorov B. I. and Peganov E. (1969) Effect of calcium, magnesium,    barium, nickel and Lanthanum ions on hyperpolarisation responses of    single nodes of Ranvier. Biofizika, 14, 474-484.-   Sokolov S. Scheuer T. and Catterall W. A. (2010) Ion permeation and    block of the gating pore in the voltage sensor of Nav1.4 channels    with hypokalemic periodic paralysis mutations. J. Gen. Physiol.,    136, 225-236.

Example 29: Guangxitoxin

GxTx-1E, a neurotoxin isolated from Plesiophrictus guangxiensis venomthat inhibits the Kv2.1 channel in pancreatic β-cells in the nanomolarrange (Herrington et al. 2006). It was hypothesised that GxTx-1Einteracts with the voltage sensors (VS) of Kv2.1 (Milescu et al. 2009,Lee et al. 2010). Guangxitoxin (GxTx-1E) was obtained from Alomone Labs.

Example 30: Demonstration that the Inward Omega Current (Iω) Through theKv2.1 R-1S/R0S/R2S Triple Mutant is Enhanced GxTx

The effects of GxTx on the Kv2.1 R-1S/R0S/R2S omega current (Iω) werestudied at a concentration above the IC50 estimated for inhibition ofthe Iα (ionic potassium outward current through the alpha pore, 100 nMGxTx-1E). FIG. 49 (A) illustrates the inward current-voltagerelationships of Iω of Kv2.1 R-1S/R0S/R2S in the absence of (squares)and after 5 minutes application of 100 nM GxTx-1E (circles). Inwardcurrents after 5 ms were normalized to the inward current at −300 mV inthe control. Normalized inward current amplitudes at the indicatedvoltages are given as mean values±S.E. FIG. 49 (B) illustrates typicalinward currents during hyperpolarising pulses through R-1S/R0S/R2S inthe absence of (left) and in the presence of 100 nM GxTx-1E (right).GxTx-1E enhanced Iω of Kv2.1 R-1S/R0S/R2S. This is the first report of adrug molecule that opens a channel omega pore. These data obtained withKv2.1 R-1S/R0S/R2S also indicate that GxTx-1E is a gating modulator thatinteracts with the voltage sensor of the Kv2.1 channel.

Example 31: Demonstration that the Inward Omega Current (Iω) Through theKv2.1 R-1S/R0S/R2S Triple Mutant is not Blocked by2-guanidinium-benzimidazole (2GBI),5-[(cyclopentylcarbonyl)amino]-2-(dimethylamino)-N-[(1R)-1-phenylethyl]-benzamide(B1), or3-methoxy-β-methyl-N-[2-(4-thiazolyl)-1H-benzimidazol-6-yl]-benzenepropanamide(RY785)

Effects on Iω on the Kv2.1 R-1 S/R0S/R2S omega current (Iω) were studiedat a concentration above the IC50 estimated for inhibition of the Iα(ionic potassium outward current through the alpha pore, 10 mM 2 GBI, 10.mu.M B1 and 10 .mu.M R785; the chemical structures of B1 and R785 aredescribed in Herrington et al. (2011) Mol. Pharm. 80:959). FIG. 50, FIG.51, and FIG. 52 illustrate, respectively, the inward current-voltagerelationships of Iω of Kv2.1 R-1S/R0S/R2S in the absence of (squares)and after 5 minutes application of 10 mM 2 GBI, 10 μM B1, or 10 μM R785(circles). Inward currents after 5 ms in control and the given compoundwere normalized to the inward current at −300 mV in the control.Normalized inward current amplitudes at the indicated voltages are givenas mean values±S.E.

REFERENCES FOR EXAMPLES 29-31

-   Herrington J, Zhou Y P, Bugianesi R M, Dulski P M, Feng Y, Warren V    A, et al. (2006) Blockers of the delayed-rectifier potassium current    in pancreatic beta-cells enhance glucose-dependent insulin    secretion. Diabetes 55(4):1034-42.-   Milescu M, Bosmans F, Lee S, Alabi A A, Kim J I, Swartz K J (2009)    Interactions between lipids and voltage sensor paddles detected with    tarantula toxins. Nat Struct Mol Biol 16(10): 1080-5.-   Lee S, Milescu M, Jung H H, Lee J Y, Bae C H, Lee C W, et al. (2010)    Solution structure of GxTX-1E, a high-affinity tarantula toxin    interacting with voltage sensors in Kv2.1 potassium channels.    Biochemistry 49(25): 5134-42.-   Herrington J, Solly K, Ratliff K S, Li N, Zhou Y P, Howard A, et    al. (2011) Identification of novel and selective Kv2 channel    inhibitors. Mol Pharmacol 80(6): 959-64.

What is claimed is:
 1. A method for identifying a compound whichmodulates the activity of a voltage-sensitive phosphatase, the methodcomprising: (a) providing a voltage-sensitive phosphatase in a polarizedstructure that separates a first medium from a second medium, whereinthe polarized structure has a voltage difference of about −300 mV toabout +300 mV, wherein the voltage-sensitive phosphatase comprises avoltage sensor domain comprising S1 to S4 hydrophobic transmembranesegments and a cytoplasmic phosphatase domain, wherein thevoltage-sensitive phosphatase exhibits altered voltage sensitivitycaused by one or more amino acid mutations in the voltage sensor domain,(b) contacting the voltage-sensitive phosphatase with a test compound,(c) determining test compound binding to the voltage-sensitivephosphatase, wherein the determining comprises: (i) measuring theactivity of the voltage-sensitive phosphatase contacted with the testcompound, (ii) measuring the activity of the voltage-sensitivephosphatase not contacted with the test compound, and (iii) comparingthe amount of said activity measured in steps (i) and (ii) wherein anincrease or decrease in the amount of activity of the voltage-sensitivephosphatase contacted with the test compound compared to thevoltage-sensitive phosphatase not contacted with the test compoundindicates that the test compound modulates the activity of thevoltage-sensitive phosphatase.
 2. The method of claim 1, wherein thestructure is a lipid bilayer.
 3. The method of claim 1, wherein thestructure is a liposome membrane.
 4. The method of claim 1, wherein thestructure comprises a naturally occurring membrane, a syntheticmembrane, or any combination thereof.
 5. The method of claim 1, whereinthe structure is a cellular membrane of a cell.
 6. The method of claim5, wherein the cell is an animal cell, a plant cell, a fungal cell, ayeast cell, a bacterial cell, or an archaebacterial cell.
 7. The methodof claim 5, wherein the cell is an oocyte, a fibroblast, an epithelialcell, or a myocyte.
 8. The method of claim 5, wherein the cell is a cellfrom a cell line.
 9. The method of claim 5, wherein the cellularmembrane is in a cell.
 10. The method of claim 5, wherein the cellularmembrane is in a permeabilized cell.
 11. The method of claim 5, whereinthe cellular membrane is not in a cell.
 12. The method of claim 5,wherein the cellular membrane comprises an extracellular membrane, anintracellular membrane, a vesicular membrane, an organelle membrane, orany combination thereof.
 13. The method of claim 1, wherein thecontacting of step (b) is performed by adding the compound to either thefirst medium or the second medium.
 14. The method of claim 1, whereinthe contacting of step (b) is performed by adding the compound to thefirst medium and the second medium.
 15. The method of claim 1, whereinthe one or more of the amino acid mutations are in the S4 hydrophobictransmembrane segment.
 16. The method of claim 1, wherein one or more ofthe amino acid mutations are not in the S4 hydrophobic transmembranesegment.
 17. The method of claim 1, wherein at least one of the aminoacid mutations is in the S4 hydrophobic transmembrane segment and atleast one of the amino acid mutations is not in the S4 hydrophobictransmembrane segment.
 18. The method of claim 1, further comprising astep of contacting the voltage sensitive phosphatase with MTSET or MTSESbefore step (b).